Indole inhibitors of MDM2 and the uses thereof

ABSTRACT

The invention relates to small molecules which function as inhibitors of the interaction between p53 and MDM2. The invention also relates to the use of these compounds for inhibiting cell growth, inducing cell death, inducing cell cycle arrest and/or sensitizing cells to additional agent(s).

The present application claims priority to U.S. Provisional PatentApplication Ser. No. 60/841,150, filed Aug. 30, 2006, which is hereinincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is in the field of medicinal chemistry. In particular,the invention relates to small molecules which function as antagonistsof the interaction between p53 and MDM2 and their use therapeutics forthe treatment of cancer and other diseases.

2. Related Art

The aggressive cancer cell phenotype is the result of a variety ofgenetic and epigenetic alterations leading to deregulation ofintracellular signaling pathways (Ponder, Nature 411:336 (2001)). Thecommonality for all cancer cells, however, is their failure to executean apoptotic program, and lack of appropriate apoptosis due to defectsin the normal apoptosis machinery is a hallmark of cancer (Lowe et al.,Carcinogenesis 21:485 (2000)). The inability of cancer cells to executean apoptotic program due to defects in the normal apoptotic machinery isthus often associated with an increase in resistance to chemotherapy,radiation, or immunotherapy-induced apoptosis. Primary or acquiredresistance of human cancer of different origins to current treatmentprotocols due to apoptosis defects is a major problem in current cancertherapy (Lowe et al., Carcinogenesis 21:485 (2000); Nicholson, Nature407:810 (2000)). Accordingly, current and future efforts towardsdesigning and developing new molecular target-specific anticancertherapies to improve survival and quality of life of cancer patientsmust include strategies that specifically target cancer cell resistanceto apoptosis. In this regard, targeting crucial negative regulators thatplay a central role in directly inhibiting apoptosis in cancer cellsrepresents a highly promising therapeutic strategy for new anticancerdrug design.

The p53 tumor suppressor plays a central role in controlling cell cycleprogression and apoptosis (Vogelstein et al., Nature 408:307 (2000)). Itis an attractive therapeutic target for anticancer drug design becauseits tumor suppressor activity can be stimulated to eradicate tumor cells(Vogelstein et al., Nature 408:307 (2000); Chene, Nat. Rev. Cancer 3:102(2003)). A new approach to stimulating the activity of p53 is throughinhibition of its interaction with the protein MDM2 using non-peptidesmall molecule inhibitors (Chene, Nat. Rev. Cancer 3:102 (2003);Vassilev et al., Science 303:844 (2004)). MDM2 and p53 are part of anauto-regulatory feed-back loop (Wu et al., Genes Dev. 7:1126 (1993)).MDM2 is transcriptionally activated by p53 and MDM2, in turn, inhibitsp53 activity by at least three mechanisms (Wu et al, Genes Dev. 7:1126(1993). First, MDM2 protein directly binds to the p53 transactivationdomain and thereby inhibits p53-mediated transactivation. Second, MDM2protein contains a nuclear export signal sequence, and upon binding top53, induces the nuclear export of p53, preventing p53 from binding tothe targeted DNAs. Third, MDM2 protein is an E3 ubiquitin ligase andupon binding to p53 is able to promote p53 degradation. Hence, byfunctioning as a potent endogenous cellular inhibitor of p53 activity,MDM2 effectively inhibits p53-mediated apoptosis, cell cycle arrest andDNA repair. Therefore, small-molecule inhibitors that bind to MDM2 andblock the interaction between MDM2 and p53 can promote the activity ofp53 in cells with a functional p53 and stimulate p53-mediated cellulareffects such as cell cycle arrest, apoptosis, or DNA repair (Chene, Nat.Rev. Cancer 3:102 (2003); Vassilev et al., Science 303:844 (2004))

Although high-affinity peptide-based inhibitors have been successfullydesigned in the past (Garcia-Echeverria et al., Med. Chem. 43:3205(2000)), these inhibitors are not drug-like molecules because of theirpoor cell permeability and in vivo bioavailability. Despite intensiveefforts by the pharmaceutical industry, high throughput screeningstrategies have had very limited success in identifying potent,non-peptide small molecule inhibitors. Accordingly, there is a need fornon-peptide, drug-like, small molecule inhibitors of the p53-MDM2interaction.

The design of non-peptide small-molecule inhibitors that target thep53-MDM2 interaction is currently being pursued as an attractivestrategy for anti-cancer drug design (Chene, Nat. Rev. Cancer 3:102(2003); Vassilev et al., Science 303:844 (2004)). The structural basisof this interaction has been established by x-ray crystallography(Kussie et al., Science 274:948 (1996)). The crystal structure showsthat the interaction between p53 and MDM2 is primarily mediated by threehydrophobic residues (Phe19, Trp23 and Leu26) from p53 and a small, deephydrophobic cleft in MDM2. This hydrophobic cleft is an ideal site fordesigning small-molecule inhibitors that can disrupt the p53-MDM2interaction (Chene, Nat. Rev. Cancer 3:102 (2003)).

SUMMARY OF THE INVENTION

It is generally accepted that the inability of cancer cells or theirsupporting cells to undergo apoptosis in response to genetic lesions orexposure to inducers of apoptosis (such as anticancer agents andradiation) is a major factor in the onset and progression of cancer. Theinduction of apoptosis in cancer cells or their supporting cells (e.g.,neovascular cells in the tumor vasculature) is thought to be a universalmechanism of action for virtually all of the effective cancertherapeutic drugs or radiation therapies on the market or in practicetoday. One reason for the inability of a cell to undergo apoptosis is adecrease in the tumor suppressor activity of p53, which in manyinstances is due to the inhibitory actions of MDM2 on p53 in tumor cellscontaining functional p53. The inhibition of p53 activity results inalterations in apoptosis pathways as well as cell cycle regulation.

The present invention contemplates that exposure of animals sufferingfrom cancer to therapeutically effective amounts of drug(s) (e.g., smallmolecules) that increase the function(s) of p53 and p53-related proteins(e.g., p63, p73) by inhibiting the interaction between p53 orp53-related proteins and MDM2 or MDM2-related proteins (e.g., MDMX) willinhibit the growth of cancer cells or supporting cells outright and/orrender such cells as a population more susceptible to the celldeath-inducing activity of cancer therapeutic drugs or radiationtherapies. In particular, the inhibitors of the invention may prolongthe half-life of p53 by interfering with the p53-MDM2 interaction thatwould normally promote degradation of p53. The present inventioncontemplates that inhibitors of the interaction between p53 orp53-related proteins and MDM2 and MDM2-related proteins satisfy an unmetneed for the treatment of multiple cancer types, either whenadministered as monotherapy to induce cell growth inhibition, apoptosisand/or cell cycle arrest in cancer cells, or when administered in atemporal relationship with additional agent(s), such as other celldeath-inducing or cell cycle disrupting cancer therapeutic drugs orradiation therapies (combination therapies), so as to render a greaterproportion of the cancer cells or supportive cells susceptible toexecuting the apoptosis program compared to the corresponding proportionof cells in an animal treated only with the cancer therapeutic drug orradiation therapy alone.

In certain embodiments of the invention, combination treatment ofanimals with a therapeutically effective amount of a compound of thepresent invention and a course of an anticancer agent or radiationproduces a greater tumor response and clinical benefit in such animalscompared to those treated with the compound or anticancerdrugs/radiation alone. Put another way, because the compounds will lowerthe apoptotic threshold of all cells, the proportion of cells that willsuccessfully execute the apoptosis program in response to the apoptosisinducing activity of anticancer drugs/radiation is increased.Alternatively, the compounds of the present invention will be used toallow administration of a lower, and therefore less toxic and moretolerable, dose of an anticancer agent and/or radiation to produce thesame tumor response/clinical benefit as the conventional dose of theanticancer agent/radiation alone. Since the doses for all approvedanticancer drugs and radiation treatments are known, the presentinvention contemplates the various combinations of them with the presentcompounds. Also, since the compounds of the present invention may act atleast in part by stimulating the pro-apoptotic and/or cellcycle-inhibiting activities of p53 and p53-related proteins, theexposure of cancer cells and supporting cells to therapeuticallyeffective amounts of the compounds should be temporally linked tocoincide with the attempts of cells to execute the apoptosis program inresponse to the anticancer agent or radiation therapy. Thus, in someembodiments, administering the compositions of the present invention inconnection with certain temporal relationships, provides especiallyefficacious therapeutic practices.

In other embodiments of the invention, inhibitors of the interactionbetween p53 or p53-related proteins and MDM2 and MDM2-related proteinsmay protect normal (e.g., non-hyperproliferative) cells from the toxiceffects of certain chemotherapeutic agents and radiation, possiblythrough the ability of the inhibitors to induce cell cycle arrest. Inparticular, the inhibitors of the invention may cause cell cycle arrestin cells comprising wild-type p53 while having no effect on cancer cellscomprising mutated or deleted p53. This differential protective effectmay allow for more effective treatment of cancer by allowing the use ofhigher doses or longer treatments of chemotherapeutic agents ortreatments without increasing the toxic side effects of such treatment.

The present invention relates to compounds that are useful forinhibiting the interaction between p53 or p53-related proteins and MDM2or MDM2-related proteins and increasing the sensitivity of cells toinducers of apoptosis and/or cell cycle arrest. In one particularembodiment, the compounds have Formula I:

or a pharmaceutically acceptable salt or prodrug thereof, wherein:

-   X is CH, O, N, or S, wherein R₈ is absent if X is O or S;-   Y is O, S, or NR′;-   R₁, R₂, R₃, R₄, R₅, R₆, and R₇ are independently H or optionally    substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl,    heterocyclic, CO₂R′, OCOR″, CONR′R″, NR″COR′, NR′SO₂R″, SO₂NR′R″,    (C═NR′)NR″R′″, or NR′R″; or-   R₇ forms an aryl, cycloalkyl, or heterocyclic group with one of R₅    or R₆;-   R₈ is H or optionally substituted alkyl, cycloalkyl, alkenyl,    cycloalkenyl, alkynyl, aryl, heterocyclic, CO₂R′, OCOR′, CONR′R″,    SO₂NR′R″, or (C═NR′)NR″R′″;-   R₉ represents a 6-chloro and a 5-fluoro group; and-   each R′, R″ and R′″ is independently H or optionally substituted    alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or    heterocyclic; or-   R′ and R″, or R″ and R′″, form a ring;    wherein one of R₃ and R₄ is CONRR′, and one of R and R′ is an    optionally substituted cycloalkyl-alkyl or    monocyclo-heterocycloalkyl group or a dihydroxyalkyl amino group not    containing a hydroxyl group at the 3-position of the alkyl group.

The invention relates to compounds represented by Formula I, which areinhibitors of the interaction between p53 or p53-related proteins andMDM2 or MDM2-related proteins. The invention relates to the use of thecompounds of the invention to induce cell cycle arrest and/or apoptosisin cells containing functional p53 or p53-related proteins. Theinvention also relates to the use of the compounds of the invention forsensitizing cells to additional agent(s), such as inducers of apoptosisand/or cell cycle arrest, and chemoprotection of normal cells throughthe induction of cell cycle arrest prior to treatment withchemotherapeutic agents. In one embodiment, the invention relates tomethods of rendering a normal cell resistant to chemotherapeutic agentsor treatments, comprising contacting the cell with a compound of theinvention. In one embodiment, the invention relates to methods ofprotecting normal cells in an animal with a hyperproliferative diseasefrom the toxic side effects of chemotherapeutic agents or treatments,comprising administering to said animal a compound of the invention. Ina particular embodiment, the invention is directed to the treatment,amelioration, or prevention of disorders, side effects, or conditionscaused by the administration of chemotherapeutic agents to normalnoncancerous cells by administering to an animal undergoing chemotherapya compound of the present invention. Examples of such disorders andconditions caused by chemotherapy include, without limitation,mucositis, stomatitis, xerostomia, gastrointestinal disorders, andalopecia.

The compounds of the invention are useful for the treatment,amelioration, or prevention of disorders, such as those responsive toinduction of apoptotic cell death, e.g., disorders characterized bydysregulation of apoptosis, including hyperproliferative diseases suchas cancer. In certain embodiments, the compounds can be used to treat,ameliorate, or prevent cancer that is characterized by resistance tocancer therapies (e.g., those cancer cells which are chemoresistant,radiation resistant, hormone resistant, and the like). In otherembodiments, the compounds can be used to treat hyperproliferativediseases characterized by expression of functional p53 or p53-relatedproteins. In other embodiments, the invention relates to the use of thecompounds of the invention to protect normal (e.g.,non-hyperproliferative) cells from the toxic side effects ofchemotherapeutic agents and treatments by the induction of cell cyclearrest in those cells.

The present invention provides pharmaceutical compositions comprising acompound of Formula I in a therapeutically effective amount to induceapoptosis in cells or to sensitize cells to inducers of apoptosis.

The invention further provides kits comprising a compound of Formula Iand instructions for administering the compound to an animal. The kitsmay optionally contain other therapeutic agents, e.g., anticancer agentsor apoptosis-modulating agents.

The invention also provides methods of making compounds of Formula I.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compounds represented by Formula I,which function as inhibitors of the interaction between p53 orp53-related proteins and MDM2 or MDM2-related proteins. By inhibitingthe negative effect of MDM2 or MDM2-related proteins on p53 orp53-related proteins, these compounds sensitize cells to inducers ofapoptosis and/or cell cycle arrest and, in some instances, themselvesinduce apoptosis and/or cell cycle arrest. Therefore, the inventionrelates to methods of sensitizing cells to inducers of apoptosis and/orcell cycle arrest and to methods of inducing apoptosis and/or cell cyclearrest in cells, comprising contacting the cells with a compound ofFormula I alone or in combination with additional agent(s), e.g., aninducer of apoptosis or a cell cycle disrupter. The invention furtherrelates to methods of treating, ameliorating, or preventing disorders inan animal, such as those that are responsive to induction of apoptosis,comprising administering to the animal a compound of Formula I andadditional agent(s), e.g., an inducer of apoptosis. Such disordersinclude those characterized by a dysregulation of apoptosis and thosecharacterized by the proliferation of cells expressing functional p53 orp53-related proteins. In other embodiments, the invention relates tomethods of protecting normal (e.g., non-hyperproliferative) cells in ananimal from the toxic side effects of chemotherapeutic agents andtreatments comprising administering to the animal a compound of FormulaI.

The terms “anticancer agent” and “anticancer drug,” as used herein,refer to any therapeutic agents (e.g., chemotherapeutic compounds and/ormolecular therapeutic compounds), antisense therapies, radiationtherapies, or surgical interventions, used in the treatment ofhyperproliferative diseases such as cancer (e.g., in mammals).

The term “prodrug,” as used herein, refers to a pharmacologicallyinactive derivative of a parent “drug” molecule that requiresbiotransformation (e.g., either spontaneous or enzymatic) within thetarget physiological system to release, or to convert (e.g.,enzymatically, physiologically, mechanically, electromagnetically) theprodrug into the active drug. Prodrugs are designed to overcome problemsassociated with stability, toxicity, lack of specificity, or limitedbioavailability. Exemplary prodrugs comprise an active drug moleculeitself and a chemical masking group (e.g., a group that reversiblysuppresses the activity of the drug). Some prodrugs are variations orderivatives of compounds that have groups cleavable under metabolicconditions. Exemplary prodrugs become pharmaceutically active in vivo orin vitro when they undergo solvolysis under physiological conditions orundergo enzymatic degradation or other biochemical transformation (e.g.,phosphorylation, hydrogenation, dehydrogenation, glycosylation).Prodrugs often offer advantages of solubility, tissue compatibility, ordelayed release in the mammalian organism. (See e.g., Bundgard, Designof Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam (1985); and Silverman,The Organic Chemistry of Drug Design and Drug Action, pp. 352-401,Academic Press, San Diego, Calif. (1992)). Common prodrugs include acidderivatives such as esters prepared by reaction of parent acids with asuitable alcohol (e.g., a lower alkanol), amides prepared by reaction ofthe parent acid compound with an amine, or basic groups reacted to forman acylated base derivative (e.g., a lower alkylamide).

The term “pharmaceutically acceptable salt,” as used herein, refers toany salt (e.g., obtained by reaction with an acid or a base) of acompound of the present invention that is physiologically tolerated inthe target animal (e.g., a mammal). Salts of the compounds of thepresent invention may be derived from inorganic or organic acids andbases. Examples of acids include, but are not limited to, hydrochloric,hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric,glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric,acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic,malonic, sulfonic, naphthalene-2-sulfonic, benzenesulfonic acid, and thelike. Other acids, such as oxalic, while not in themselvespharmaceutically acceptable, may be employed in the preparation of saltsuseful as intermediates in obtaining the compounds of the invention andtheir pharmaceutically acceptable acid addition salts.

Examples of bases include, but are not limited to, alkali metal (e.g.,sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides,ammonia, and compounds of formula NW₄ ⁺, wherein W is C₁₋₄ alkyl, andthe like.

Examples of salts include, but are not limited to: acetate, adipate,alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, chloride, bromide,iodide, 2-hydroxyethanesulfonate, lactate, maleate, mesylate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate,pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like.Other examples of salts include anions of the compounds of the presentinvention compounded with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄⁺ (wherein W is a C₁₋₄ alkyl group), and the like. For therapeutic use,salts of the compounds of the present invention are contemplated asbeing pharmaceutically acceptable. However, salts of acids and basesthat are non-pharmaceutically acceptable may also find use, for example,in the preparation or purification of a pharmaceutically acceptablecompound.

The term “therapeutically effective amount,” as used herein, refers tothat amount of the therapeutic agent sufficient to result inamelioration of one or more symptoms of a disorder, or preventadvancement of a disorder, or cause regression of the disorder. Forexample, with respect to the treatment of cancer, in one embodiment, atherapeutically effective amount will refer to the amount of atherapeutic agent that decreases the rate of tumor growth, decreasestumor mass, decreases the number of metastases, increases time to tumorprogression, or increases survival time by at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 100%.

The terms “sensitize” and “sensitizing,” as used herein, refer tomaking, through the administration of a first agent (e.g., a compound ofFormula I), an animal or a cell within an animal more susceptible, ormore responsive, to the biological effects (e.g., promotion orretardation of an aspect of cellular function including, but not limitedto, cell division, cell growth, proliferation, invasion, angiogenesis,necrosis, or apoptosis) of a second agent. The sensitizing effect of afirst agent on a target cell can be measured as the difference in theintended biological effect (e.g., promotion or retardation of an aspectof cellular function including, but not limited to, cell growth,proliferation, invasion, angiogenesis, or apoptosis) observed upon theadministration of a second agent with and without administration of thefirst agent. The response of the sensitized cell can be increased by atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 100%, atleast 150%, at least 200%, at least 350%, at least 300%, at least 350%,at least 400%, at least 450%, or at least 500% over the response in theabsence of the first agent.

The term “dysregulation of apoptosis,” as used herein, refers to anyaberration in the ability of (e.g., predisposition) a cell to undergocell death via apoptosis. Dysregulation of apoptosis is associated withor induced by a variety of conditions, non-limiting examples of whichinclude, autoimmune disorders (e.g., systemic lupus erythematosus,rheumatoid arthritis, graft-versus-host disease, myasthenia gravis, orSjögren's syndrome), chronic inflammatory conditions (e.g., psoriasis,asthma or Crohn's disease), hyperproliferative disorders (e.g., tumors,B cell lymphomas, or T cell lymphomas), viral infections (e.g., herpes,papilloma, or HIV), and other conditions such as osteoarthritis andatherosclerosis. It should be noted that when the dysregulation isinduced by or associated with a viral infection, the viral infection mayor may not be detectable at the time dysregulation occurs or isobserved. That is, viral-induced dysregulation can occur even after thedisappearance of symptoms of viral infection.

The term “functional p53,” as used herein, refers to wild-type p53expressed at normal, high, or low levels and mutant p53 that retains atleast 5% of the activity of wild-type p53, e.g., at least 10%, 20%, 30%,40%, 50%, or more of wild-type activity.

The term “p53-related protein,” as used herein, refers to proteins thathave at least 25% sequence homology with p53, have tumor suppressoractivity, and are inhibited by interaction with MDM2 or MDM2-relatedproteins. Examples of p53-related proteins include, but are not limitedto, p63 and p73.

The term “MDM2-related protein,” as used herein, refers to proteins thathave at least 25% sequence homology with MDM2, and interact with andinhibit p53 or p53-related proteins. Examples of MDM2-related proteinsinclude, but are not limited to, MDMX and HDM2.

The term “hyperproliferative disease,” as used herein, refers to anycondition in which a localized population of proliferating cells in ananimal is not governed by the usual limitations of normal growth.Examples of hyperproliferative disorders include tumors, neoplasms,lymphomas and the like. A neoplasm is said to be benign if it does notundergo invasion or metastasis and malignant if it does either of these.A “metastatic” cell means that the cell can invade and destroyneighboring body structures. Hyperplasia is a form of cell proliferationinvolving an increase in cell number in a tissue or organ withoutsignificant alteration in structure or function. Metaplasia is a form ofcontrolled cell growth in which one type of fully differentiated cellsubstitutes for another type of differentiated cell.

The pathological growth of activated lymphoid cells often results in anautoimmune disorder or a chronic inflammatory condition. As used herein,the term “autoimmune disorder” refers to any condition in which anorganism produces antibodies or immune cells which recognize theorganism's own molecules, cells or tissues. Non-limiting examples ofautoimmune disorders include autoimmune hemolytic anemia, autoimmunehepatitis, Berger's disease or IgA nephropathy, celiac sprue, chronicfatigue syndrome, Crohn's disease, dermatomyositis, fibromyalgia, graftversus host disease, Grave's disease, Hashimoto's thyroiditis,idiopathic thrombocytopenia purpura, lichen planus, multiple sclerosis,myasthenia gravis, psoriasis, rheumatic fever, rheumatic arthritis,scleroderma, Sjögren's syndrome, systemic lupus erythematosus, type 1diabetes, ulcerative colitis, vitiligo, and the like.

The term “neoplastic disease,” as used herein, refers to any abnormalgrowth of cells being either benign (non-cancerous) or malignant(cancerous).

The term “normal cell,” as used herein, refers to a cell that is notundergoing abnormal growth or division. Normal cells are non-cancerousand are not part of any hyperproliferative disease or disorder.

The term “anti-neoplastic agent,” as used herein, refers to any compoundthat retards the proliferation, growth, or spread of a targeted (e.g.,malignant) neoplasm.

The terms “prevent,” “preventing,” and “prevention,” as used herein,refer to a decrease in the occurrence of pathological cells (e.g.,hyperproliferative or neoplastic cells) in an animal. The prevention maybe complete, e.g., the total absence of pathological cells in a subject.The prevention may also be partial, such that the occurrence ofpathological cells in a subject is less than that which would haveoccurred without the present invention.

The term “apoptosis-modulating agents,” as used herein, refers to agentswhich are involved in modulating (e.g., inhibiting, decreasing,increasing, promoting) apoptosis. Examples of apoptosis-modulatingagents include proteins which comprise a death domain such as, but notlimited to, Fas/CD95, TRAMP, TNF RI, DR1, DR2, DR3, DR4, DR5, DR6, FADD,and RIP. Other examples of apoptosis-modulating agents include, but arenot limited to, TNFα, Fas ligand, antibodies to Fas/CD95 and other TNFfamily receptors, TRAIL (also known as Apo2 Ligand or Apo2L/TRAIL),antibodies to TRAIL-R1 or TRAIL-R2, Bcl-2, p53, BAX, BAD, Akt, CAD, PI3kinase, PP1, and caspase proteins. Modulating agents broadly includeagonists and antagonists of TNF family receptors and TNF family ligands.Apoptosis-modulating agents may be soluble or membrane bound (e.g.ligand or receptor). Apoptosis-modulating agents include those which areinducers of apoptosis, such as TNF or a TNF-related ligand, particularlya TRAMP ligand, a Fas/CD95 ligand, a TNFR-1 ligand, or TRAIL.

In one aspect of the invention, the inhibitors of the interactionbetween p53 and MDM2 are compounds of Formula I:

or a pharmaceutically acceptable salt or prodrug thereof, wherein:

-   X is CH, O, N, or S, wherein R₈ is absent if X is O or S;-   Y is O, S, or NR′;-   R₁, R₂, R₃, R₄, R₅, R₆, and R₇ are independently H or optionally    substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl,    heterocyclic, CO₂R′, OCOR′, CONR′R″, NR″COR′, NR′SO₂R″, SO₂NR′R″,    (C═NR′)NR″R′″, or NR′R″; or-   R₇ forms an aryl, cycloalkyl, or heterocyclic group with one of R₅    or R₆;-   R₈ is H or optionally substituted alkyl, cycloalkyl, alkenyl,    cycloalkenyl, alkynyl, aryl, heterocyclic, CO₂R′, OCOR′, CONR′R″,    SO₂NR′R″, or (C═NR′)NR″R′″;-   R₉ represents a 6-chloro and a 5-fluoro group; and-   each R′, R″ and R′″ is independently H or optionally substituted    alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or    heterocyclic; or-   R′ and R″, or R″ and R′″, form a ring;    wherein one of R₃ and R₄ is CONRR′, and one of R and R′ is an    optionally substituted cycloalkyl-alkyl or    monocyclo-heterocycloalkyl group or a dihydroxyalkyl amino group not    containing a hydroxyl group at the 3-position of the alkyl group.

In a more particular embodiment, one of R₁ and R₂ of Formula I is asubstituted or unsubstituted aryl (e.g., phenyl), substituted orunsubstituted heteroaryl, cycloalkyl, straight or branched alkyl, amideor ester.

In another embodiment, one of R₅ and R₆ is a C₃₋₁₈ alkyl group, e.g.,propyl, isopropyl, sec-butyl, tert-butyl, isopentyl, cyclopentyl,norbornyl, or adamantyl, or a 5- or 6-membered aryl or heteroaryl group.

In another embodiment, the compounds of Formula I have a stereochemicalstructure as shown in Formula II or Formula III:

or a pharmaceutically acceptable salt or prodrug thereof.

In one embodiment, the compounds of Formula I have Formula IV:

wherein R₁-R₉ and Y are as defined above.

In another embodiment, the compounds of Formula IV have a stereochemicalstructure as shown in Formula V or Formula VI:

or a pharmaceutically acceptable salt or prodrug thereof.

In one embodiment, the compounds of Formula I have Formula VII:

wherein R₁-R₉ are as defined above.

In another embodiment, the compounds of Formula VII have astereochemical structure as shown in Formula VIII or Formula IX:

or a pharmaceutically acceptable salt or prodrug thereof.

In one embodiment, the compounds of Formula I have Formula X:

wherein:

-   R₁, R₅, R₇, and R₉ are as defined above;-   R₁₀ is H; and-   R₁₁ is an optionally substituted cycloalkyl-alkyl or    monocyclo-heterocycloalkyl group or a dihydroxyalkyl amino group not    containing a hydroxyl group at the 3-position of the alkyl group;-   or R₁₀ and R₁₁ together form an optionally substituted    monocyclo-heterocycloalkyl group.

Examples of substituted cycloalkyl-alkyl groups include C₁₋₆ alkylsubstituted by a cycloalkyl group as described herein below which issubstituted further by one or more hydroxyl groups. Examples ofheterocycloalkyl groups include C₁₋₆ alkyl substituted by amonocyclo-heterocycloalkyl group as described herein below.

Particular examples of cycloalkyl-alkyl groups include2-(3-hydroxycyclopentyl)ethylamino, and3-(3-hydroxycyclopentyl)propylamino groups. The respective hydroxylgroups may have either the R or S configurations.

Particular examples of substituted heterocycloalkyl groups include2-(1-morpholinyl)ethylamino and 3-(1-morpholinyl)propylamino groups.

Particular examples of dihydroxyalkylamino groups include R and S4,5-dihydroxypentylamino, and 4-hydroxy-3-(methylhydroxy)butylaminogroups.

In a further embodiment, the compounds of Formula I have one of FormulaeXI-XXVI:

wherein R₁, R₅, R₇, R₉, R₁₀, and R₁₁ are as defined above.

In another embodiment, the compounds of Formula I have one of FormulaeXXVII and XXVIII:

wherein R₁, R₅, R₇, R₉, R₁₀, and R₁₁ are as defined above.

In another embodiment, the compounds of Formula I have Formula XLVIII:

wherein:

-   R₁, R₃, R₄, R₅ and R₉ are as defined above.

In a further embodiment, the compounds of Formula I have one of FormulaeXLIX-LXIV:

wherein:

-   R₁, R₃, R₄, R₅ and R₉ are as defined above.

In a further embodiment, the compounds of Formula I have Formula LXV:

wherein:

-   R₁, R₄, R₅, R₉, R₁₀, and R₁₁ are as defined above.

In a further embodiment, the compounds of Formula I have one of FormulaeLXVI and LXVII:

wherein:

-   R₁, R₄, R₅, R₉, R₁₀, and R₁₁ are as defined above.

Particular embodiments of the present invention include, withoutlimitation, any one of the following compounds:

Another particular embodiment of the present invention includes, withoutlimitation, any one of the following compounds:

Useful alkyl groups include straight-chained or branched C₁₋₁₈ alkylgroups, especially methyl, ethyl, propyl, isopropyl, t-butyl, sec-butyl,3-pentyl, adamantyl, norbornyl, and 3-hexyl groups.

Useful alkenyl groups include straight-chained or branched C₂₋₁₈ alkylgroups, especially ethenyl, propenyl, isopropenyl, butenyl, isobutenyl,and hexenyl.

Useful alkynyl groups are C₂₋₁₈ alkynyl groups, especially ethynyl,propynyl, butynyl, and 2-butynyl groups

Useful cycloalkyl groups are C₃₋₈ cycloalkyl. Typical cycloalkyl groupsinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl andcycloheptyl.

Useful aryl groups include C₆₋₁₄ aryl, especially phenyl, naphthyl,phenanthrenyl, anthracenyl, indenyl, azulenyl, biphenyl, biphenylenyl,and fluorenyl groups.

Useful heteroaryl groups include thienyl, benzo[b]thienyl,naphtho[2,3-b]thienyl, thianthrenyl, furyl, pyranyl, isobenzofuranyl,chromenyl, xanthenyl, phenoxanthenyl, 2H-pyrrolyl, pyrrolyl, imidazolyl,pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl,isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl,isoquinolyl, quinolyl, phthalzinyl, naphthyridinyl, quinozalinyl,cinnolinyl, pteridinyl, carbazolyl, β-carbolinyl, phenanthridinyl,acridinyl, perimidinyl, phenanthrolinyl, phenazinyl, isothiazolyl,phenothiazinyl, isoxazolyl, furazanyl, phenoxazinyl,1,4-dihydroquinoxaline-2,3-dione, 7-aminoisocoumarin,pyrido[1,2-a]pyrimidin-4-one, 1,2-benzoisoxazol-3-yl, benzimidazolyl,2-oxindolyl, and 2-oxobenzimidazolyl. Where the heteroaryl groupcontains a nitrogen atom in a ring, such nitrogen atom may be in theform of an N-oxide, e.g., a pyridyl N-oxide, pyrazinyl N-oxide,pyrimidinyl N-oxide, and the like.

Useful heterocyclic groups include monocyclic heterocyclic groups suchas tetrahydrofuranyl, pyranyl, piperidinyl, piperizinyl, pyrrolidinyl,imidazolidinyl, imidazolinyl, morpholinyl, pyrazolidinyl, pyrazolinyl,and the like. Multi-cyclo heterocyclic groups include indolinyl,isoindolinyl, quinuclidinyl, chromanyl, isochromanyl, tetronoyl,tetrahydroisoquinolinyl groups, as well as heterocyclic groups fusedwith a heteroaryl ring, e.g. optionally substituted5,6-dihydro-8H-[1,2,4]triazolo[4,3-A]pyrazinyl groups.

Optional substituents include one or more alkyl; halo; haloalkyl;cycloalkyl; aryl optionally substituted with one or more lower alkyl,lower alkoxy, methylenedioxy, halo, haloalkyl, aminosulfonyl, aryl, orheteroaryl groups; aryloxy optionally substituted with one or more loweralkyl, lower alkoxy, methylenedioxy, halo, haloalkyl, aminosulfonyl,aryl, or heteroaryl groups; aralkyl; heteroaryl optionally substitutedwith one or more lower alkyl, lower alkoxy, methylenedioxy, halo,haloalkyl, aminosulfonyl, aryl, or heteroaryl groups; heteroaryloxyoptionally substituted with one or more lower alkyl, lower alkoxy,methylenedioxy, halo, haloalkyl, aminosulfonyl, aryl, or heteroarylgroups; alkoxy; alkylthio; arylthio; amido; amino; aminoalkyl,alkylamino, acyloxy; arylacyloxy optionally substituted with one or morelower alkyl, lower alkoxy, methylenedioxy, halo, haloalkyl,aminosulfonyl, aryl, or heteroaryl groups; diphenylphosphinyloxyoptionally substituted with one or more lower alkyl, lower alkoxy,methylenedioxy, halo, haloalkyl, aminosulfonyl, aryl, or heteroarylgroups; heterocyclo optionally substituted with one or more lower alkyl,lower alkoxy, methylenedioxy, halo, haloalkyl, aminosulfonyl, aryl,heteroaryl, amino acid substituted sulfonyl, or amino acid derivativesubstituted sulfonyl groups; heterocycloacyl optionally substituted withone or more lower alkyl, lower alkoxy, methylenedioxy, halo, haloalkyl,aminosulfonyl, aryl, or heteroaryl groups; heterocycloalkoxy optionallysubstituted with one or more lower alkyl, lower alkoxy, methylenedioxy,halo, haloalkyl, aminosulfonyl, aryl, or heteroaryl groups; partiallyunsaturated heterocycloalkyl optionally substituted with one or morelower alkyl, lower alkoxy, methylenedioxy, halo, haloalkyl,aminosulfonyl, aryl, or heteroaryl groups; or partially unsaturatedheterocycloalkyloxy optionally substituted with one or more lower alkyl,lower alkoxy, methylenedioxy, halo, haloalkyl, aminosulfonyl, aryl, orheteroaryl groups.

Certain of the compounds of the present invention may exist asstereoisomers including optical isomers. The invention includes allstereoisomers, both as pure individual stereoisomer preparations andenriched preparations of each, and both the racemic mixtures of suchstereoisomers as well as the individual enantiomers that may beseparated according to methods that are well known to those of skill inthe art.

The compounds and processes of the present invention will be betterunderstood in connection with the following synthetic schemes whichillustrate the methods by which the compounds of the invention may beprepared. Starting materials can be obtained from commercial sources orprepared by well-established literature methods known to those ofordinary skill in the art. It will be readily apparent to one ofordinary skill in the art that the compounds defined above can besynthesized by substitution of the appropriate reagents and agents inthe syntheses shown below.

Compounds have the general structure of formula X are synthesized byusing a asymmetric 1,3-dipolar cycloaddition as the key step (Scheme 1).

Reagents and conditions: a) CH₂Cl₂—CH₃CN, KF—Al₂O₃, microwave, ormethanol, piperidine reflux; b) 4 Å molecular sieves, toluene, 70° C.;c) amine, r.t.; d) Pb(OAc)₄, CH₂Cl₂-MeOH (1:1), 0° C., or ammoniumcerium(IV) nitrate (CAN), CH₃CN, K₂CO₃, r.t.

Compounds having Formula LXV are prepared by a similar method as thepreparation of Formula X (Scheme 5).

Reagents and conditions: a) CH₂Cl₂—CH₃CN, KF—Al₂O₃, microwave, ormethanol, piperidine reflux; b) 4 Å molecular sieves, toluene, 70° C.;c) amine, r.t.; d) Pb(OAc)₄, CH₂Cl₂-MeOH (1:1), 0° C., or ammoniumcerium(IV) nitrate (CAN), CH₃CN, K₂CO₃, r.t.

One aspect of the invention related to methods of preparing MDM2inhibitor compounds. In one embodiment, the invention relates to amethod of preparing a compound having formula X, comprising

-   a) condensing a compound of Formula 1 with a compound of Formula 2,    e.g., in a solvent or a mixture of solvents (e.g., CH₂Cl₂ and CH₃CN)    under microwave in the presence of a catalyst (e.g., KF—Al₂O₃) or in    the presence of a base in a suitable solvent to form a compound of    Formula 3;

-   b) condensing the compound of Formula 3 with a compound of Formula 4    and a compound of Formula 5, e.g., in a non-polar solvent (e.g.,    toluene) in the presence of a dehydrating agent (e.g., 4 Å molecular    sieve) at elevated temperature (e.g., about 70° C.) to form a    compound of Formula 6; and

-   c) treating the compound of Formula 6 with an oxidizing agent (e.g.,    Pb(OAc)₄, Ammonium cerium nitrate) in a solvent or mixture of    solvents (e.g., CH₂Cl₂ and MeOH, CH₃CN) at a suitable temperature    (e.g., about 0° C. or room temperature) to form a compound of    Formula X.

In another embodiment, the invention relates to a method of preparing acompound having formula LXV, comprising

-   a) condensing a compound of Formula 16 with a compound of Formula 2,    e.g., in a solvent or a mixture of solvents (e.g., CH₂Cl₂ and CH₃CN)    under microwave in the presence of a catalyst (e.g., KF—Al₂O₃) or in    the presence of a base in a suitable solvent to form a compound of    Formula 17;

-   b) condensing the compound of Formula 17 with a compound of Formula    4 and a compound of Formula 5, e.g., in a non-polar solvent (e.g.,    toluene) in the presence of a dehydrating agent (e.g., 4 Å molecular    sieve) at elevated temperature (e.g., about 70° C.) to form a    compound of Formula 18; and

-   c) treating the compound of Formula 18 with an oxidizing agent    (e.g., Pb(OAc)₄, Ammonium cerium nitrate) in a solvent or mixture of    solvents (e.g., CH₂Cl₂ and MeOH, CH₃CN) at a suitable temperature    (e.g., about 0° C. or room temperature) to form a compound of    Formula LXV;

wherein:

-   R₁, R₅, R₉, R₁₀ and R₁₁ are as defined above.

An important aspect of the present invention is that compounds ofFormula I induce cell cycle arrest and/or apoptosis and also potentiatethe induction of cell cycle arrest and/or apoptosis either alone or inresponse to additional apoptosis induction signals. Therefore, it iscontemplated that these compounds sensitize cells to induction of cellcycle arrest and/or apoptosis, including cells that are resistant tosuch inducing stimuli. The inhibitors of the interaction between p53 orp53-related proteins and MDM2 or MDM2-related proteins of the presentinvention can be used to induce apoptosis in any disorder that can betreated, ameliorated, or prevented by the induction of apoptosis. In oneembodiment, the inhibitors can be used to induce apoptosis in cellscomprising functional p53 or p53-related proteins.

In another embodiment, the invention pertains to modulating an apoptosisassociated state which is associated with one or moreapoptosis-modulating agents. Examples of apoptosis-modulating agentsinclude, but are not limited to, Fas/CD95, TRAMP, TNF RI, DR1, DR2, DR3,DR4, DR5, DR6, FADD, RIP, TNFα, Fas ligand, TRAIL, antibodies toTRAIL-R1 or TRAIL-R2, Bcl-2, p53, BAX, BAD, Akt, CAD, PI3 kinase, PP1,and caspase proteins. Other agents involved in the initiation, decisionand degradation phase of apoptosis are also included. Examples ofapoptosis-modulating agents include agents, the activity, presence, orchange in concentration of which, can modulate apoptosis in a subject.Apoptosis-modulating agents include those which are inducers ofapoptosis, such as TNF or a TNF-related ligand, particularly a TRAMPligand, a Fas/CD95 ligand, a TNFR-1 ligand, or TRAIL.

In some embodiments, the compositions and methods of the presentinvention are used to treat diseased cells, tissues, organs, orpathological conditions and/or disease states in an animal (e.g., amammalian subject including, but not limited to, humans and veterinaryanimals). In this regard, various diseases and pathologies are amenableto treatment or prophylaxis using the present methods and compositions.A non-limiting exemplary list of these diseases and conditions includes,but is not limited to, breast cancer, prostate cancer, lymphoma, skincancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma,ovarian cancer, brain cancer, primary brain carcinoma, head-neck cancer,glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lungcancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma,lung carcinoma, small-cell lung carcinoma, Wilms' tumor, cervicalcarcinoma, testicular carcinoma, bladder carcinoma, pancreaticcarcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma,genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma,myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma,endometrial carcinoma, adrenal cortex carcinoma, malignant pancreaticinsulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosisfungoides, malignant hypercalcemia, cervical hyperplasia, leukemia,acute lymphocytic leukemia, chronic lymphocytic leukemia, acutemyelogenous leukemia, chronic myelogenous leukemia, chronic granulocyticleukemia, acute granulocytic leukemia, hairy cell leukemia,neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera,essential thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma,soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, andretinoblastoma, and the like, T and B cell mediated autoimmune diseases;inflammatory diseases; infections; hyperproliferative diseases; AIDS;degenerative conditions, vascular diseases, and the like. In someembodiments, the cancer cells being treated are metastatic. In otherembodiments, the cancer cells being treated are resistant to anticanceragents.

In some embodiments, infections suitable for treatment with thecompositions and methods of the present invention include, but are notlimited to, infections caused by viruses, bacteria, fungi, mycoplasma,prions, and the like.

Some embodiments of the present invention provide methods foradministering an effective amount of a compound of Formula I and atleast one additional therapeutic agent (including, but not limited to,chemotherapeutic antineoplastics, apoptosis-modulating agents,antimicrobials, antivirals, antifungals, and anti-inflammatory agents)and/or therapeutic technique (e.g., surgical intervention, and/orradiotherapies).

A number of suitable anticancer agents are contemplated for use in themethods of the present invention. Indeed, the present inventioncontemplates, but is not limited to, administration of numerousanticancer agents such as: agents that induce apoptosis; polynucleotides(e.g., anti-sense, ribozymes, siRNA); polypeptides (e.g., enzymes andantibodies); biological mimetics (e.g., gossypol or BH3 mimetics);agents that bind (e.g., oligomerize or complex) with a Bcl-2 familyprotein such as Bax; alkaloids; alkylating agents; antitumorantibiotics; antimetabolites; hormones; platinum compounds; monoclonalor polyclonal antibodies (e.g., antibodies conjugated with anticancerdrugs, toxins, defensins), toxins; radionuclides; biological responsemodifiers (e.g., interferons (e.g., IFN-α) and interleukins (e.g.,IL-2)); adoptive immunotherapy agents; hematopoietic growth factors;agents that induce tumor cell differentiation (e.g., all-trans-retinoicacid); gene therapy reagents (e.g., antisense therapy reagents andnucleotides); tumor vaccines; angiogenesis inhibitors; proteosomeinhibitors: NF-κB modulators; anti-CDK compounds; HDAC inhibitors; andthe like. Numerous other examples of chemotherapeutic compounds andanticancer therapies suitable for co-administration with the disclosedcompounds are known to those skilled in the art.

In certain embodiments, anticancer agents comprise agents that induce orstimulate apoptosis. Agents that induce apoptosis include, but are notlimited to, radiation (e.g., X-rays, gamma rays, UV); tumor necrosisfactor (TNF)-related factors (e.g., TNF family receptor proteins, TNFfamily ligands, TRAIL, antibodies to TRAIL-R1 or TRAIL-R2); kinaseinhibitors (e.g., epidermal growth factor receptor (EGFR) kinaseinhibitor, vascular growth factor receptor (VGFR) kinase inhibitor,fibroblast growth factor receptor (FGFR) kinase inhibitor,platelet-derived growth factor receptor (PDGFR) kinase inhibitor, andBcr-Abl kinase inhibitors (such as GLEEVEC)); antisense molecules;antibodies (e.g., HERCEPTIN, RITUXAN, ZEVALIN, and AVASTIN);anti-estrogens (e.g., raloxifene and tamoxifen); anti-androgens (e.g.,flutamide, bicalutamide, finasteride, aminoglutethamide, ketoconazole,and corticosteroids); cyclooxygenase 2 (COX-2) inhibitors (e.g.,celecoxib, meloxicam, NS-398, and non-steroidal anti-inflammatory drugs(NSAIDs)); anti-inflammatory drugs (e.g., butazolidin, DECADRON,DELTASONE, dexamethasone, dexamethasone intensol, DEXONE, HEXADROL,hydroxychloroquine, METICORTEN, ORADEXON, ORASONE, oxyphenbutazone,PEDIAPRED, phenylbutazone, PLAQUENIL, prednisolone, prednisone, PRELONE,and TANDEARIL); and cancer chemotherapeutic drugs (e.g., irinotecan(CAMPTOSAR), CPT-11, fludarabine (FLUDARA), dacarbazine (DTIC),dexamethasone, mitoxantrone, MYLOTARG, VP-16, cisplatin, carboplatin,oxaliplatin, 5-FU, doxorubicin, gemcitabine, bortezomib, gefitinib,bevacizumab, TAXOTERE or TAXOL); cellular signaling molecules; ceramidesand cytokines; staurosporine, and the like.

In still other embodiments, the compositions and methods of the presentinvention provide a compound of Formula I and at least oneanti-hyperproliferative or antineoplastic agent selected from alkylatingagents, antimetabolites, and natural products (e.g., herbs and otherplant and/or animal derived compounds).

Alkylating agents suitable for use in the present compositions andmethods include, but are not limited to: 1) nitrogen mustards (e.g.,mechlorethamine, cyclophosphamide, ifosfamide, melphalan (L-sarcolysin);and chlorambucil); 2) ethylenimines and methylmelamines (e.g.,hexamethylmelamine and thiotepa); 3) alkyl sulfonates (e.g., busulfan);4) nitrosoureas (e.g., carmustine (BCNU); lomustine (CCNU); semustine(methyl-CCNU); and streptozocin (streptozotocin)); and 5) triazenes(e.g., dacarbazine (DTIC; dimethyltriazenoimid-azolecarboxamide).

In some embodiments, antimetabolites suitable for use in the presentcompositions and methods include, but are not limited to: 1) folic acidanalogs (e.g., methotrexate (amethopterin)); 2) pyrimidine analogs(e.g., fluorouracil (5-fluorouracil; 5-FU), floxuridine(fluorode-oxyuridine; FudR), and cytarabine (cytosine arabinoside)); and3) purine analogs (e.g., mercaptopurine (6-mercaptopurine; 6-MP),thioguanine (6-thioguanine; TG), and pentostatin (2′-deoxycoformycin)).

In still further embodiments, chemotherapeutic agents suitable for usein the compositions and methods of the present invention include, butare not limited to: 1) vinca alkaloids (e.g., vinblastine (VLB),vincristine); 2) epipodophyllotoxins (e.g., etoposide and teniposide);3) antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin(daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin(mithramycin), and mitomycin (mitomycin C)); 4) enzymes (e.g.,L-asparaginase); 5) biological response modifiers (e.g.,interferon-alfa); 6) platinum coordinating complexes (e.g., cisplatin(cis-DDP) and carboplatin); 7) anthracenediones (e.g., mitoxantrone); 8)substituted ureas (e.g., hydroxyurea); 9) methylhydrazine derivatives(e.g., procarbazine (N-methylhydrazine; MIH)); 10) adrenocorticalsuppressants (e.g., mitotane (o,p′-DDD) and aminoglutethimide); 11)adrenocorticosteroids (e.g., prednisone); 12) progestins (e.g.,hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrolacetate); 13) estrogens (e.g., diethylstilbestrol and ethinylestradiol); 14) antiestrogens (e.g., tamoxifen); 15) androgens (e.g.,testosterone propionate and fluoxymesterone); 16) antiandrogens (e.g.,flutamide): and 17) gonadotropin-releasing hormone analogs (e.g.,leuprolide).

Any oncolytic agent that is routinely used in a cancer therapy contextfinds use in the compositions and methods of the present invention. Forexample, the U.S. Food and Drug Administration maintains a formulary ofoncolytic agents approved for use in the United States. Internationalcounterpart agencies to the U.S.F.D.A. maintain similar formularies.Table 1 provides a list of exemplary antineoplastic agents approved foruse in the U.S. Those skilled in the art will appreciate that the“product labels” required on all U.S. approved chemotherapeuticsdescribe approved indications, dosing information, toxicity data, andthe like, for the exemplary agents.

TABLE 1 Aldesleukin Proleukin Chiron Corp., Emeryville, (des-alanyl-1,serine-125 human interleukin-2) CA Alemtuzumab Campath Millennium andILEX (IgG1κ anti CD52 antibody) Partners, LP, Cambridge, MA AlitretinoinPanretin Ligand Pharmaceuticals, Inc., (9-cis-retinoic acid) San DiegoCA Allopurinol Zyloprim GlaxoSmithKline, Research(1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4- Triangle Park, NC onemonosodium salt) Altretamine Hexalen US Bioscience, West(N,N,N′,N′,N″,N″,-hexamethyl-1,3,5-triazine- Conshohocken, PA2,4,6-triamine) Amifostine Ethyol US Bioscience (ethanethiol,2-[(3-aminopropyl)amino]-, dihydrogen phosphate (ester)) AnastrozoleArimidex AstraZeneca (1,3-Benzenediacetonitrile, a,a,a′,a′-Pharmaceuticals, LP, tetramethyl-5-(1H-1,2,4-triazol-1-ylmethyl))Wilmington, DE Arsenic trioxide Trisenox Cell Therapeutic, Inc.,Seattle, WA Asparaginase Elspar Merck & Co., Inc., (L-asparagineamidohydrolase, type EC-2) Whitehouse Station, NJ BCG Live TICE BCGOrganon Teknika, Corp., (lyophilized preparation of an attenuated strainDurham, NC of Mycobacterium bovis (Bacillus Calmette- Gukin [BCG],substrain Montreal) bexarotene capsules Targretin Ligand Pharmaceuticals(4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-napthalenyl)ethenyl]benzoic acid) bexarotene gel TargretinLigand Pharmaceuticals Bleomycin Blenoxane Bristol-Myers Squibb Co.,(cytotoxic glycopeptide antibiotics produced NY, NY by Streptomycesverticillus; bleomycin A₂ and bleomycin B₂) Capecitabine Xeloda Roche(5′-deoxy-5-fluoro-N-[(pentyloxy)carbonyl]- cytidine) CarboplatinParaplatin Bristol-Myers Squibb (platinum, diammine [1,1-cyclobutanedicarboxylato(2-)-0,0′]-,(SP-4-2)) Carmustine BCNU,Bristol-Myers Squibb (1,3-bis(2-chloroethyl)-1-nitrosourea) BiCNUCarmustine with Polifeprosan 20 Implant Gliadel GuilfordPharmaceuticals, Wafer Inc., Baltimore, MD Celecoxib Celebrex SearlePharmaceuticals, (as 4-[5-(4-methylphenyl)-3-(trifluoromethyl)- England1H-pyrazol-1-yl] benzenesulfonamide) Chlorambucil LeukeranGlaxoSmithKline (4-[bis(2chlorethyl)amino]benzenebutanoic acid)Cisplatin Platinol Bristol-Myers Squibb (PtCl₂H₆N₂) CladribineLeustatin, R. W. Johnson (2-chloro-2′-deoxy-b-D-adenosine) 2-CdAPharmaceutical Research Institute, Raritan, NJ Cyclophosphamide Cytoxan,Bristol-Myers Squibb (2-[bis(2-chloroethyl)amino]tetrahydro-2H- Neosar13,2-oxazaphosphorine 2-oxide monohydrate) Cytarabine Cytosar-UPharmacia & Upjohn (1-b-D-Arabinofuranosylcytosine, C₉H₁₃N₃O₅) Companycytarabine liposomal DepoCyt Skye Pharmaceuticals, Inc., San Diego, CADacarbazine DTIC- Bayer AG, Leverkusen,(5-(3,3-dimethyl-1-triazeno)-imidazole-4- Dome Germany carboxamide(DTIC)) Dactinomycin, actinomycin D Cosmegen Merck (actinomycin producedby Streptomyces parvullus, C₆₂H₈₆N₁₂O₁₆) Darbepoetin alfa Aranesp Amgen,Inc., Thousand Oaks, (recombinant peptide) CA daunorubicin liposomalDanuoXome Nexstar Pharmaceuticals,((8S-cis)-8-acetyl-10-[(3-amino-2,3,6-trideoxy- Inc., Boulder, COá-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12- naphthacenedionehydrochloride) Daunorubicin HCl, daunomycin Cerubidine Wyeth Ayerst,Madison, NJ ((1S,3S)-3-Acetyl-1,2,3,4,6,11-hexahydro-3,5,12-trihydroxy-10-methoxy-6,11-dioxo-1- naphthacenyl3-amino-2,3,6-trideoxy-(alpha)- L-lyxo-hexopyranoside hydrochloride)Denileukin diftitox Ontak Seragen, Inc., Hopkinton, (recombinantpeptide) MA Dexrazoxane Zinecard Pharmacia & Upjohn((S)-4,4′-(1-methyl-1,2-ethanediyl)bis-2,6- Company piperazinedione)Docetaxel Taxotere Aventis Pharmaceuticals,((2R,3S)-N-carboxy-3-phenylisoserine, N-tert- Inc., Bridgewater, NJbutyl ester, 13-ester with 5b-20-epoxy-12a,4,7b,10b,13a-hexahydroxytax-11-en-9- one 4-acetate 2-benzoate,trihydrate) Doxorubicin HCl Adriamycin, Pharmacia & Upjohn(8S,10S)-10-[(3-amino-2,3,6-trideoxy-a-L- Rubex Companylyxo-hexopyranosyl)oxy]-8-glycolyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12- naphthacenedionehydrochloride) doxorubicin Adriamycin Pharmacia & Upjohn PFS CompanyIntravenous injection doxorubicin liposomal Doxil SequusPharmaceuticals, Inc., Menlo park, CA dromostanolone propionateDromostanolone Eli Lilly & Company,(17b-Hydroxy-2a-methyl-5a-androstan-3-one Indianapolis, IN propionate)dromostanolone propionate Masterone Syntex, Corp., Palo Alto, CAinjection Elliott's B Solution Elliott's B Orphan Medical, Inc SolutionEpirubicin Ellence Pharmacia & Upjohn((8S-cis)-10-[(3-amino-2,3,6-trideoxy-a-L- Companyarabino-hexopyranosyl)oxy]-7,8,9,10- tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12- naphthacenedione hydrochloride) Epoetinalfa Epogen Amgen, Inc (recombinant peptide) Estramustine EmcytPharmacia & Upjohn (estra-1,3,5(10)-triene-3,17-diol(17(beta))-, 3-Company [bis(2-chloroethyl)carbamate]17-(dihydrogen phosphate), disodiumsalt, monohydrate, or estradiol 3-[bis(2-chloroethyl)carbamate]17-(dihydrogen phosphate), disodium salt, monohydrate) Etoposide phosphateEtopophos Bristol-Myers Squibb (4′-Demethylepipodophyllotoxin9-[4,6-O-(R)- ethylidene-(beta)-D-glucopyranoside], 4′- (dihydrogenphosphate)) etoposide, VP-16 Vepesid Bristol-Myers Squibb(4′-demethylepipodophyllotoxin 9-[4,6-0-(R)-ethylidene-(beta)-D-glucopyranoside]) Exemestane Aromasin Pharmacia &Upjohn (6-methylenandrosta-1,4-diene-3,17-dione) Company FilgrastimNeupogen Amgen, Inc (r-metHuG-CSF) floxuridine (intraarterial) FUDRRoche (2′-deoxy-5-fluorouridine) Fludarabine Fludara BerlexLaboratories, Inc., (fluorinated nucleotide analog of the antiviralCedar Knolls, NJ agent vidarabine, 9-b-D- arabinofuranosyladenine(ara-A)) Fluorouracil, 5-FU Adrucil ICN Pharmaceuticals, Inc.,(5-fluoro-2,4(1H,3H)-pyrimidinedione) Humacao, Puerto Rico FulvestrantFaslodex IPR Pharmaceuticals, (7-alpha-[9-(4,4,5,5,5-penta Guayama,Puerto Rico fluoropentylsulphinyl)nonyl]estra-1,3,5-(10)-triene-3,17-beta-diol) Gemcitabine Gemzar Eli Lilly(2′-deoxy-2′,2′-difluorocytidine monohydrochloride (b-isomer))Gemtuzumab Ozogamicin Mylotarg Wyeth Ayerst (anti-CD33 hP67.6) Goserelinacetate Zoladex AstraZeneca Pharmaceuticals (acetate salt of[D-Ser(But)⁶,Azgly¹⁰]LHRH; Implantpyro-Glu-His-Trp-Ser-Tyr-D-Ser(But)-Leu- Arg-Pro-Azgly-NH2 acetate[C₅₉H₈₄N₁₈O₁₄•(C₂H₄O₂)_(x) Hydroxyurea Hydrea Bristol-Myers SquibbIbritumomab Tiuxetan Zevalin Biogen IDEC, Inc., (immunoconjugateresulting from a thiourea Cambridge MA covalent bond between themonoclonal antibody Ibritumomab and the linker-chelator tiuxetan[N-[2-bis(carboxymethyl)amino]-3-(p-isothiocyanatophenyl)-propyl]-[N-[2-bis(carboxymethyl)amino]-2-(methyl)- ethyl]glycine) Idarubicin IdamycinPharmacia & Upjohn (5,12-Naphthacenedione, 9-acetyl-7-[(3- Companyamino-2,3,6-trideoxy-(alpha)-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro- 6,9,11-trihydroxyhydrochloride,(7S-cis)) Ifosfamide IFEX Bristol-Myers Squibb (3-(2-chloroethyl)-2-[(2-chloroethyl)amino]tetrahydro-2H-1,3,2- oxazaphosphorine 2-oxide)Imatinib Mesilate Gleevec Novartis AG, Basel,(4-[(4-Methyl-1-piperazinyl)methyl]-N-[4- Switzerlandmethyl-3-[[4-(3-pyridinyl)-2- pyrimidinyl]amino]-phenyl]benzamidemethanesulfonate) Interferon alfa-2a Roferon-A Hoffmann-La Roche, Inc.,(recombinant peptide) Nutley, NJ Interferon alfa-2b Intron A ScheringAG, Berlin, (recombinant peptide) (Lyophilized Germany Betaseron)Irinotecan HCl Camptosar Pharmacia & Upjohn((4S)-4,11-diethyl-4-hydroxy-9-[(4-piperi- Companydinopiperidino)carbonyloxy]-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H) dione hydrochloridetrihydrate) Letrozole Femara Novartis(4,4′-(1H-1,2,4-Triazol-1-ylmethylene) dibenzonitrile) LeucovorinWellcovorin, Immunex, Corp., Seattle, WA (L-Glutamic acid,N[4[[(2amino-5-formyl- Leucovorin 1,4,5,6,7,8 hexahydro4oxo6-pteridinyl)methyl]amino]benzoyl], calcium salt (1:1)) Levamisole HClErgamisol Janssen Research ((−)-(S)-2,3,5,6-tetrahydro-6-phenylimidazoFoundation, Titusville, NJ [2,1-b]thiazole monohydrochlorideC₁₁H₁₂N₂S•HCl) Lomustine CeeNU Bristol-Myers Squibb(1-(2-chloro-ethyl)-3-cyclohexyl-1- nitrosourea) Meclorethamine,nitrogen mustard Mustargen Merck (2-chloro-N-(2-chloroethyl)-N-methylethanamine hydrochloride) Megestrol acetate Megace Bristol-MyersSquibb 17α(acetyloxy)-6-methylpregna-4,6-diene- 3,20-dione Melphalan,L-PAM Alkeran GlaxoSmithKline(4-[bis(2-chloroethyl)amino]-L-phenylalanine) Mercaptopurine, 6-MPPurinethol GlaxoSmithKline (1,7-dihydro-6H-purine-6-thione monohydrate)Mesna Mesnex Asta Medica (sodium 2-mercaptoethane sulfonate)Methotrexate Methotrexate Lederle Laboratories (N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L- glutamic acid) MethoxsalenUvadex Therakos, Inc., Way Exton,(9-methoxy-7H-furo[3,2-g][1]-benzopyran-7- Pa one) Mitomycin C MutamycinBristol-Myers Squibb mitomycin C Mitozytrex SuperGen, Inc., Dublin, CAMitotane Lysodren Bristol-Myers Squibb(1,1-dichloro-2-(o-chlorophenyl)-2-(p- chlorophenyl)ethane) MitoxantroneNovantrone Immunex Corporation (1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9,10- anthracenedione dihydrochloride)Nandrolone phenpropionate Durabolin- Organon, Inc., West Orange, 50 NJNofetumomab Verluma Boehringer Ingelheim Pharma KG, Germany OprelvekinNeumega Genetics Institute, Inc., (IL-11) Alexandria, VA OxaliplatinEloxatin Sanofi Synthelabo, Inc., NY,(cis-[(1R,2R)-1,2-cyclohexanediamine-N,N′] NY[oxalato(2-)-O,O′]platinum) Paclitaxel TAXOL Bristol-Myers Squibb(5β,20-Epoxy-1,2a,4,7β,10β,13a- hexahydroxytax-11-en-9-one4,10-diacetate 2- benzoate 13-ester with (2R,3S)-N-benzoyl-3-phenylisoserine) Pamidronate Aredia Novartis (phosphonic acid(3-amino-1- hydroxypropylidene)bis-, disodium salt, pentahydrate, (APD))Pegademase Adagen Enzon Pharmaceuticals, Inc., ((monomethoxypolyethyleneglycol (Pegademase Bridgewater, NJ succinimidyl)11-17-adenosinedeaminase) Bovine) Pegaspargase Oncaspar Enzon (monomethoxypolyethyleneglycol succinimidyl L-asparaginase) Pegfilgrastim Neulasta Amgen, Inc(covalent conjugate of recombinant methionyl human G-CSF (Filgrastim)and monomethoxypolyethylene glycol) Pentostatin Nipent Parke-DavisPharmaceutical Co., Rockville, MD Pipobroman Vercyte AbbottLaboratories, Abbott Park, IL Plicamycin, Mithramycin Mithracin Pfizer,Inc., NY, NY (antibiotic produced by Streptomyces plicatus) Porfimersodium Photofrin QLT Phototherapeutics, Inc., Vancouver, CanadaProcarbazine Matulane Sigma Tau Pharmaceuticals,(N-isopropyl-μ-(2-methylhydrazino)-p- Inc., Gaithersburg, MD toluamidemonohydrochloride) Quinacrine Atabrine Abbott Labs(6-chloro-9-(1-methyl-4-diethyl-amine) butylamino-2-methoxyacridine)Rasburicase Elitek Sanofi-Synthelabo, Inc., (recombinant peptide)Rituximab Rituxan Genentech, Inc., South San (recombinant anti-CD20antibody) Francisco, CA Sargramostim Prokine Immunex Corp (recombinantpeptide) Streptozocin Zanosar Pharmacia & Upjohn (streptozocin2-deoxy-2- Company [[(methylnitrosoamino)carbonyl]amino]- a(andb)-D-glucopyranose and 220 mg citric acid anhydrous) Talc SclerosolBryan, Corp., Woburn, MA (Mg₃Si₄O₁₀ (OH)₂) Tamoxifen NolvadexAstraZeneca Pharmaceuticals ((Z)2-[4-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylethanamine 2-hydroxy-1,2,3- propanetricarboxylate (1:1))Temozolomide Temodar Schering (3,4-dihydro-3-methyl-4-oxoimidazo[5,1-d]-as-tetrazine-8-carboxamide) teniposide, VM-26 Vumon Bristol-Myers Squibb(4′-demethylepipodophyllotoxin 9-[4,6-0-(R)-2-thenylidene-(beta)-D-glucopyranoside]) Testolactone TeslacBristol-Myers Squibb (13-hydroxy-3-oxo-13,17-secoandrosta-1,4-dien-17-oic acid [dgr]-lactone) Thioguanine, 6-TG ThioguanineGlaxoSmithKline (2-amino-1,7-dihydro-6H-purine-6-thione) ThiotepaThioplex Immunex Corporation (Aziridine,1,1′,1″-phosphinothioylidynetris-, or Tris (1-aziridinyl)phosphinesulfide) Topotecan HCl Hycamtin GlaxoSmithKline((S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14-(4H,12H)-dione monohydrochloride) ToremifeneFareston Roberts Pharmaceutical(2-(p-[(Z)-4-chloro-1,2-diphenyl-1-butenyl]- Corp., Eatontown, NJphenoxy)-N,N-dimethylethylamine citrate (1:1)) Tositumomab, I 131Tositumomab Bexxar Corixa Corp., Seattle, WA (recombinant murineimmunotherapeutic monoclonal IgG_(2a) lambda anti-CD20 antibody (I 131is a radioimmunotherapeutic antibody)) Trastuzumab Herceptin Genentech,Inc (recombinant monoclonal IgG₁ kappa anti- HER2 antibody) Tretinoin,ATRA Vesanoid Roche (all-trans retinoic acid) Uracil Mustard UracilRoberts Labs Mustard Capsules Valrubicin,N-trifluoroacetyladriamycin-14- Valstar Anthra --> Medeva valerate((2S-cis)-2-[1,2,3,4,6,11-hexahydro-2,5,12- trihydroxy-7methoxy-6,11-dioxo-[[4 2,3,6-trideoxy-3-[(trifluoroacetyl)-amino-α-L-lyxo-hexopyranosyl]oxyl]-2-naphthacenyl]-2- oxoethyl pentanoate) Vinblastine,Leurocristine Velban Eli Lilly (C₄₆H₅₆N₄₀O₁₀•H₂SO₄) Vincristine OncovinEli Lilly (C₄₆H₅₆N₄₀O₁₀•H₂SO₄) Vinorelbine Navelbine GlaxoSmithKline(3′,4′-didehydro-4′-deoxy-C′- norvincaleukoblastine [R-(R*,R*)-2,3-dihydroxybutanedioate (1:2)(salt)]) Zoledronate, Zoledronic acid ZometaNovartis ((1-Hydroxy-2-imidazol-1-yl-phosphonoethyl) phosphonic acidmonohydrate)

Anticancer agents further include compounds which have been identifiedto have anticancer activity but are not currently approved by the U.S.Food and Drug Administration or other counterpart agencies or areundergoing evaluation for new uses. Examples include, but are notlimited to, 3-AP, 12-O-tetradecanoylphorbol-13-acetate, 17AAG, 852A,ABI-007, ABR-217620, ABT-751, ADI-PEG 20, AE-941, AG-013736, AGRO100,alanosine, AMG 706, antibody G250, antineoplastons, AP23573, apaziquone,APC8015, atiprimod, ATN-161, atrasenten, azacitidine, BB-10901,BCX-1777, bevacizumab, BG00001, bicalutamide, BMS 247550, bortezomib,bryostatin-1, buserelin, calcitriol, CCI-779, CDB-2914, cefixime,cetuximab, CG0070, cilengitide, clofarabine, combretastatin A4phosphate, CP-675,206, CP-724,714, CpG 7909, curcumin, decitabine,DENSPM, doxercalciferol, E7070, E7389, ecteinascidin 743, efaproxiral,eflomithine, EKB-569, enzastaurin, erlotinib, exisulind, fenretinide,flavopiridol, fludarabine, flutamide, fotemustine, FR901228, G17DT,galiximab, gefitinib, genistein, glufosfamide, GTI-2040, histrelin,HKI-272, homoharringtonine, HSPPC-96, hu14.18-interleukin-2 fusionprotein, HuMax-CD4, iloprost, imiquimod, infliximab, interleukin-12,IPI-504, irofulven, ixabepilone, lapatinib, lenalidomide, lestaurtinib,leuprolide, LMB-9 immunotoxin, lonafarnib, luniliximab, mafosfamide,MB07133, MDX-010, MLN2704, monoclonal antibody 3F8, monoclonal antibodyJ591, motexafin, MS-275, MVA-MUC1-IL2, nilutamide, nitrocamptothecin,nolatrexed dihydrochloride, nolvadex, NS-9, O6-benzylguanine, oblimersensodium, ONYX-015, oregovomab, OSI-774, panitumumab, paraplatin,PD-0325901, pemetrexed, PHY906, pioglitazone, pirfenidone, pixantrone,PS-341, PSC 833, PXD101, pyrazoloacridine, R115777, RAD001, ranpirnase,rebeccamycin analogue, rhuAngiostatin protein, rhuMab 2C4,rosiglitazone, rubitecan, S-1, S-8184, satraplatin, SB-, 15992,SGN-0010, SGN-40, sorafenib, SR31747A, ST1571, SU011248, suberoylanilidehydroxamic acid, suramin, talabostat, talampanel, tariquidar,temsirolimus, TGFa-PE38 immunotoxin, thalidomide, thymalfasin,tipifarnib, tirapazamine, TLK286, trabectedin, trimetrexate glucuronate,TroVax, UCN-1, valproic acid, vinflunine, VNP40101M, volociximab,vorinostat, VX-680, ZD1839, ZD6474, zileuton, and zosuquidartrihydrochloride.

For a more detailed description of anticancer agents and othertherapeutic agents, those skilled in the art are referred to any numberof instructive manuals including, but not limited to, the Physician'sDesk Reference and to Goodman and Gilman's “Pharmaceutical Basis ofTherapeutics” tenth edition, Eds. Hardman et al., 2002.

The present invention provides methods for administering a compound ofFormula I with radiation therapy. The invention is not limited by thetypes, amounts, or delivery and administration systems used to deliverthe therapeutic dose of radiation to an animal. For example, the animalmay receive photon radiotherapy, particle beam radiation therapy, othertypes of radiotherapies, and combinations thereof. In some embodiments,the radiation is delivered to the animal using a linear accelerator. Instill other embodiments, the radiation is delivered using a gamma knife.

The source of radiation can be external or internal to the animal.External radiation therapy is most common and involves directing a beamof high-energy radiation to a tumor site through the skin using, forinstance, a linear accelerator. While the beam of radiation is localizedto the tumor site, it is nearly impossible to avoid exposure of normal,healthy tissue. However, external radiation is usually well tolerated byanimals. Internal radiation therapy involves implanting aradiation-emitting source, such as beads, wires, pellets, capsules,particles, and the like, inside the body at or near the tumor siteincluding the use of delivery systems that specifically target cancercells (e.g., using particles attached to cancer cell binding ligands).Such implants can be removed following treatment, or left in the bodyinactive. Types of internal radiation therapy include, but are notlimited to, brachytherapy, interstitial irradiation, intracavityirradiation, radioimmunotherapy, and the like.

The animal may optionally receive radiosensitizers (e.g., metronidazole,misonidazole, intra-arterial Budr, intravenous iododeoxyuridine (IudR),nitroimidazole, 5-substituted-4-nitroimidazoles, 2H-isoindolediones,[[(2-bromoethyl)-amino]methyl]-nitro-1H-imidazole-1-ethanol,nitroaniline derivatives, DNA-affinic hypoxia selective cytotoxins,halogenated DNA ligand, 1,2,4 benzotriazine oxides, 2-nitroimidazolederivatives, fluorine-containing nitroazole derivatives, benzamide,nicotinamide, acridine-intercalator, 5-thiotretrazole derivative,3-nitro-1,2,4-triazole, 4,5-dinitroimidazole derivative, hydroxylatedtexaphrins, cisplatin, mitomycin, tiripazamine, nitrosourea,mercaptopurine, methotrexate, fluorouracil, bleomycin, vincristine,carboplatin, epirubicin, doxorubicin, cyclophosphamide, vindesine,etoposide, paclitaxel, heat (hyperthermia), and the like),radioprotectors (e.g., cysteamine, aminoalkyl dihydrogenphosphorothioates, amifostine (WR 2721), IL-1, IL-6, and the like).Radiosensitizers enhance the killing of tumor cells. Radioprotectorsprotect healthy tissue from the harmful effects of radiation.

Any type of radiation can be administered to an animal, so long as thedose of radiation is tolerated by the animal without unacceptablenegative side-effects. Suitable types of radiotherapy include, forexample, ionizing (electromagnetic) radiotherapy (e.g., X-rays or gammarays) or particle beam radiation therapy (e.g., high linear energyradiation). Ionizing radiation is defined as radiation comprisingparticles or photons that have sufficient energy to produce ionization,i.e., gain or loss of electrons (as described in, for example, U.S. Pat.No. 5,770,581 incorporated herein by reference in its entirety). Theeffects of radiation can be at least partially controlled by theclinician. In one embodiment, the dose of radiation is fractionated formaximal target cell exposure and reduced toxicity.

In one embodiment, the total dose of radiation administered to an animalis about 0.01 Gray (Gy) to about 100 Gy. In another embodiment, about 10Gy to about 65 Gy (e.g., about 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy,45 Gy, 50 Gy, 55 Gy, or 60 Gy) are administered over the course oftreatment. While in some embodiments a complete dose of radiation can beadministered over the course of one day, the total dose is ideallyfractionated and administered over several days. Desirably, radiotherapyis administered over the course of at least about 3 days, e.g., at least5, 7, 10, 14, 17, 21, 25, 28, 32, 35, 38, 42, 46, 52, or 56 days (about1-8 weeks). Accordingly, a daily dose of radiation will compriseapproximately 1-5 Gy (e.g., about 1 Gy, 1.5 Gy, 1.8 Gy, 2 Gy, 2.5 Gy,2.8 Gy, 3 Gy, 3.2 Gy, 3.5 Gy, 3.8 Gy, 4 Gy, 4.2 Gy, or 4.5 Gy), or 1-2Gy (e.g., 1.5-2 Gy). The daily dose of radiation should be sufficient toinduce destruction of the targeted cells. If stretched over a period, inone embodiment, radiation is not administered every day, therebyallowing the animal to rest and the effects of the therapy to berealized. For example, radiation desirably is administered on 5consecutive days, and not administered on 2 days, for each week oftreatment, thereby allowing 2 days of rest per week. However, radiationcan be administered 1 day/week, 2 days/week, 3 days/week, 4 days/week, 5days/week, 6 days/week, or all 7 days/week, depending on the animal'sresponsiveness and any potential side effects. Radiation therapy can beinitiated at any time in the therapeutic period. In one embodiment,radiation is initiated in week 1 or week 2, and is administered for theremaining duration of the therapeutic period. For example, radiation isadministered in weeks 1-6 or in weeks 2-6 of a therapeutic periodcomprising 6 weeks for treating, for instance, a solid tumor.Alternatively, radiation is administered in weeks 1-5 or weeks 2-5 of atherapeutic period comprising 5 weeks. These exemplary radiotherapyadministration schedules are not intended, however, to limit the presentinvention.

Antimicrobial therapeutic agents may also be used as therapeutic agentsin the present invention. Any agent that can kill, inhibit, or otherwiseattenuate the function of microbial organisms may be used, as well asany agent contemplated to have such activities. Antimicrobial agentsinclude, but are not limited to, natural and synthetic antibiotics,antibodies, inhibitory proteins (e.g., defensins), antisense nucleicacids, membrane disruptive agents and the like, used alone or incombination. Indeed, any type of antibiotic may be used including, butnot limited to, antibacterial agents, antiviral agents, antifungalagents, and the like.

In some embodiments of the present invention, a compound of Formula Iand one or more therapeutic agents or anticancer agents are administeredto an animal under one or more of the following conditions: at differentperiodicities, at different durations, at different concentrations, bydifferent administration routes, etc. In some embodiments, the compoundis administered prior to the therapeutic or anticancer agent, e.g., 0.5,1, 2, 3, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2,3, or 4 weeks prior to the administration of the therapeutic oranticancer agent. In some embodiments, the compound is administeredafter the therapeutic or anticancer agent, e.g., 0.5, 1, 2, 3, 4, 5, 10,12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks afterthe administration of the anticancer agent. In some embodiments, thecompound and the therapeutic or anticancer agent are administeredconcurrently but on different schedules, e.g., the compound isadministered daily while the therapeutic or anticancer agent isadministered once a week, once every two weeks, once every three weeks,or once every four weeks. In other embodiments, the compound isadministered once a week while the therapeutic or anticancer agent isadministered daily, once a week, once every two weeks, once every threeweeks, or once every four weeks.

Compositions within the scope of this invention include all compositionswherein the compounds of the present invention are contained in anamount which is effective to achieve its intended purpose. Whileindividual needs vary, determination of optimal ranges of effectiveamounts of each component is within the skill of the art. Typically, thecompounds may be administered to mammals, e.g. humans, orally at a doseof 0.0025 to 50 mg/kg, or an equivalent amount of the pharmaceuticallyacceptable salt thereof, per day of the body weight of the mammal beingtreated for disorders responsive to induction of apoptosis. In oneembodiment, about 0.01 to about 25 mg/kg is orally administered totreat, ameliorate, or prevent such disorders. For intramuscularinjection, the dose is generally about one-half of the oral dose. Forexample, a suitable intramuscular dose would be about 0.0025 to about 25mg/kg, or from about 0.01 to about 5 mg/kg.

The unit oral dose may comprise from about 0.01 to about 1000 mg, forexample, about 0.1 to about 100 mg of the compound. The unit dose may beadministered one or more times daily as one or more tablets or capsuleseach containing from about 0.1 to about 10 mg, conveniently about 0.25to 50 mg of the compound or its solvates.

In a topical formulation, the compound may be present at a concentrationof about 0.01 to 100 mg per gram of carrier. In a one embodiment, thecompound is present at a concentration of about 0.07-1.0 mg/ml, forexample, about 0.1-0.5 mg/ml, and in one embodiment, about 0.4 mg/ml.

In addition to administering the compound as a raw chemical, thecompounds of the invention may be administered as part of apharmaceutical preparation containing suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the compounds into preparations which can beused pharmaceutically. The preparations, particularly those preparationswhich can be administered orally or topically and which can be used forone type of administration, such as tablets, dragees, slow releaselozenges and capsules, mouth rinses and mouth washes, gels, liquidsuspensions, hair rinses, hair gels, shampoos and also preparationswhich can be administered rectally, such as suppositories, as well assuitable solutions for administration by intravenous infusion,injection, topically or orally, contain from about 0.01 to 99 percent,in one embodiment from about 0.25 to 75 percent of active compound(s),together with the excipient.

The pharmaceutical compositions of the invention may be administered toany animal which may experience the beneficial effects of the compoundsof the invention. Foremost among such animals are mammals, e.g., humans,although the invention is not intended to be so limited. Other animalsinclude veterinary animals (cows, sheep, pigs, horses, dogs, cats andthe like).

The compounds and pharmaceutical compositions thereof may beadministered by any means that achieve their intended purpose. Forexample, administration may be by parenteral, subcutaneous, intravenous,intramuscular, intraperitoneal, transdermal, buccal, intrathecal,intracranial, intranasal or topical routes. Alternatively, orconcurrently, administration may be by the oral route. The dosageadministered will be dependent upon the age, health, and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the effect desired.

The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, for example, by means ofconventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral usecan be obtained by combining the active compounds with solid excipients,optionally grinding the resulting mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

Suitable excipients are, in particular, fillers such as saccharides, forexample lactose or sucrose, mannitol or sorbitol, cellulose preparationsand/or calcium phosphates, for example tricalcium phosphate or calciumhydrogen phosphate, as well as binders such as starch paste, using, forexample, maize starch, wheat starch, rice starch, potato starch,gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose,sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired,disintegrating agents may be added such as the above-mentioned starchesand also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar,or alginic acid or a salt thereof, such as sodium alginate. Auxiliariesare, above all, flow-regulating agents and lubricants, for example,silica, talc, stearic acid or salts thereof, such as magnesium stearateor calcium stearate, and/or polyethylene glycol. Dragee cores areprovided with suitable coatings which, if desired, are resistant togastric juices. For this purpose, concentrated saccharide solutions maybe used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquersolutions and suitable organic solvents or solvent mixtures. In order toproduce coatings resistant to gastric juices, solutions of suitablecellulose preparations such as acetylcellulose phthalate orhydroxypropylmethyl-cellulose phthalate, are used. Dye stuffs orpigments may be added to the tablets or dragee coatings, for example,for identification or in order to characterize combinations of activecompound doses.

Other pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer such as glycerol or sorbitol. The push-fitcapsules can contain the active compounds in the form of granules whichmay be mixed with fillers such as lactose, binders such as starches,and/or lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds are in oneembodiment dissolved or suspended in suitable liquids, such as fattyoils, or liquid paraffin. In addition, stabilizers may be added.

Possible pharmaceutical preparations which can be used rectally include,for example, suppositories, which consist of a combination of one ormore of the active compounds with a suppository base. Suitablesuppository bases are, for example, natural or synthetic triglycerides,or paraffin hydrocarbons. In addition, it is also possible to usegelatin rectal capsules which consist of a combination of the activecompounds with a base. Possible base materials include, for example,liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form, for example,water-soluble salts and alkaline solutions. In addition, suspensions ofthe active compounds as appropriate oily injection suspensions may beadministered. Suitable lipophilic solvents or vehicles include fattyoils, for example, sesame oil, or synthetic fatty acid esters, forexample, ethyl oleate or triglycerides or polyethylene glycol-400.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension include, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran. Optionally, the suspension may alsocontain stabilizers.

The topical compositions of this invention are formulated in oneembodiment as oils, creams, lotions, ointments and the like by choice ofappropriate carriers. Suitable carriers include vegetable or mineraloils, white petrolatum (white soft paraffin), branched chain fats oroils, animal fats and high molecular weight alcohol (greater than C₁₂).The carriers may be those in which the active ingredient is soluble.Emulsifiers, stabilizers, humectants and antioxidants may also beincluded as well as agents imparting color or fragrance, if desired.Additionally, transdermal penetration enhancers can be employed in thesetopical formulations. Examples of such enhancers can be found in U.S.Pat. Nos. 3,989,816 and 4,444,762.

Creams may be formulated from a mixture of mineral oil, self-emulsifyingbeeswax and water in which mixture the active ingredient, dissolved in asmall amount of an oil such as almond oil, is admixed. A typical exampleof such a cream is one which includes about 40 parts water, about 20parts beeswax, about 40 parts mineral oil and about 1 part almond oil.

Ointments may be formulated by mixing a solution of the activeingredient in a vegetable oil such as almond oil with warm soft paraffinand allowing the mixture to cool. A typical example of such an ointmentis one which includes about 30% almond oil and about 70% white softparaffin by weight.

Lotions may be conveniently prepared by dissolving the activeingredient, in a suitable high molecular weight alcohol such aspropylene glycol or polyethylene glycol.

The following examples are illustrative, but not limiting, of the methodand compositions of the present invention. Other suitable modificationsand adaptations of the variety of conditions and parameters normallyencountered in clinical therapy and which are obvious to those skilledin the art are within the spirit and scope of the invention.

EXAMPLE 16-CHLORO-4′-(3-CHLORO-PHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID (3,4-DIHYDROXY-BUTYL)-AMIDE

¹H NMR (300 MHz, CD₃OH), δ 7.70 (d, 1H, J=8.4 Hz), 7.40-7.20 (m, 3H),7.09 (m, 1H), 6.88 (d, 1H, J=6.0 Hz), 5.22 (d, 1H, J=11.4 Hz), 4.39 (m,1H), 4.11 (d, 1H, J=11.1 Hz), 3.65-3.32 (m, 9H), 1.85 (m, 1H), 1.60 (m,1H), 1.41 (m, 1H), 1.19 (m, 1H), 0.84 (s, 9H).

EXAMPLE 26-CHLORO-4′-(3-CHLORO-PHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID (2,3-DIHYDROXY-PROPYL)-AMIDE

¹H NMR (300 MHz, CD₃OH), δ 7.74 (d, 1H, J=7.5 Hz), 7.35-7.15 (m, 3H),7.11 (m, 1H), 6.88 (d, 1H, J=6.0 Hz), 5.22 (d, 1H, J=11.4 Hz), 4.33 (m,1H), 4.28 (d, 1H, J=10.5 Hz), 3.67 (m, 2H), 3.65-3.32 (m, 4H), 2.05 (m,1H), 1.90 (m, 1H), 1.56 (m, 1H), 0.87 (s, 9H).

EXAMPLE 36-CHLORO-4′-(3-CHLORO-PHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID (3,5-DIHYDROXY-PENTYL)-AMIDE

¹H NMR (300 MHz, CD₃OH), δ 7.72 (d, 1H, J=8.4 Hz), 7.35-7.15 (m, 3H),7.10 (m, 1H), 6.88 (d, 1H, J=6.0 Hz), 5.23 (d, 1H, J=11.1 Hz), 4.65 (m,1H), 4.13 (d, 1H, J=11.4 Hz), 3.61 (m, 2H), 3.65-3.32 (m, 5H), 2.02 (m,1H), 1.93 (m, 1H), 1.56 (m, 3H), 1.15 (m, 1H), 0.92 (s, 9H).

EXAMPLE 46-CHLORO-4′-(3-CHLORO-PHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID [2-(3-HYDROXY-CYCLOPENTYL)-ETHYL]-AMIDE

¹H NMR (300 MHz, CD₃OH), δ 7.69 (d, 1H, J=8.4 Hz), 7.40-7.20 (m, 3H),7.10 (m, 1H), 6.86 (m, 1H), 5.24 (d, 1H, J=11.1 Hz), 4.35 (d, 1H, J=8.4Hz), 4.09 (m, 1H), 3.61-3.32 (m, 2H), 2.96 (m, 1H), 2.21-1.12 (m, 11H),0.85 (s, 9H).

EXAMPLE 56-CHLORO-4′-(3-CHLORO-PHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID [2-(3-HYDROXY-CYCLOPENTYL)-ETHYL]-AMIDE

¹H NMR (300 MHz, CD₃OH), δ 7.69 (d, 1H, J=8.4 Hz), 7.40-7.20 (m, 3H),7.10 (m, 1H), 6.86 (m, 1H), 5.24 (d, 1H, J=11.1 Hz), 4.48 (d, 1H, J=8.4Hz), 4.12 (m, 1H), 3.64-3.32 (m, 2H), 2.96 (m, 1H), 2.20-1.10 (m, 1H),0.85 (s, 9H).

EXAMPLE 66-CHLORO-4′-(3-CHLORO-PHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID [2-(3-HYDROXY-CYCLOPENTYL)-ETHYL]-AMIDE

¹H NMR (300 MHz, CD₃OH), δ 7.69 (d, 1H, J=8.4 Hz), 7.40-7.20 (m, 3H),7.10 (m, 1H), 6.86 (m, 1H), 5.24 (d, 1H, J=11.1 Hz), 4.47 (d, 1H, J=8.4Hz), 4.11 (m, 1H), 3.65-3.32 (m, 2H), 2.96 (m, 1H), 2.20-1.10 (m, 1H),0.91 (s, 9H).

EXAMPLE 76-CHLORO-4′-(3-CHLORO-PHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID [2-(3-HYDROXY-CYCLOPENTYL)-ETHYL]-AMIDE

¹H NMR (300 MHz, CD₃OH), δ 7.72 (d, 1H, J=8.4 Hz), 7.38-7.22 (m, 3H),7.10 (m, 1H), 6.87 (m, 1H), 5.23 (d, 1H, J=11.1 Hz), 4.48 (d, 1H, J=8.4Hz), 4.12 (m, 1H), 3.56-3.25 (m, 2H), 2.98 (m, 1H), 2.20-1.10 (m, 11H),0.86 (s, 9H).

EXAMPLE 86-CHLORO-4′-(3-CHLORO-4-FLUORO-PHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID (4,5-DIHYDROXY-PENTYL)-AMIDE

¹H NMR (CD₃OD, 300 MHz), δ 7.73 (d, J=8.4 Hz, 1H), 7.41 (d, J=7.0 Hz,1H), 7.14 (d, J=6.7 Hz, 2H), 5.25 (d, J=11.3 Hz, 1H), 4.49 (d, J=6.6 Hz,1H), 4.15 (d, J=6.4 Hz, 1H), 3.48-3.10 (m, 5H), 1.94 (dd, J=8.1, 15.4Hz, 1H), 1.70-1.10 (m, 5H), 0.92 (s, 9H).

EXAMPLE 96-CHLORO-4′-(3-CHLORO-4-FLUORO-PHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID (3-MORPHOLIN-4-YL-PROPYL)-AMIDE

¹H NMR (CD₃OD, 300 MHz), δ 7.82-7.19 (m, 4H), 6.90 (d, J=5.56, 1H), 5.39(d, J=9.9 Hz, 1H), 4.55 (d, J=6.9 Hz, 1H), 4.28 (d, J=9.3 Hz, 1H),4.20-3.85 (m, 4H), 3.70-3.40 (m, 2H), 3.25-3.00 (m, 4H), 2.20-1.85 (m,3H), 1.69 (d, J=15.3 Hz, 2H), 0.934 (s, 9H), 0.70 (t, J=11.3 Hz, 1H).

EXAMPLE 106-CHLORO-4′-(3-CHLORO-2-FLUORO-PHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID (4-HYDROXY-3-HYDROXYMETHYL-BUTYL)-AMIDE

H¹ (300 MHz CD₃OD), δ 8.56 (s br, 1H), 7.68 (t, J=6.6 Hz, 1H), 7.42 (t,J=7.2 Hz, 1H), 7.25 (t, J=7.8 Hz, 1H), 7.05 (d, J=8.7 Hz, 1H), 6.95 (d,J=6.0 Hz, 1H), 5.22 (d, J=10.5 Hz, 1H), 4.54 (d, J=10.5 Hz, 1H),4.34-4.31 (m, 1H), 3.51-3.49 (m, 4H), 3.36-3.32 (m, 2H), 2.08 (dd,J=15.3, 7.5 Hz, 1H), 1.53-1.49 (m, 2H), 1.33 (dd, J=15.3, 2.7 Hz, 1H),0.93-0.89 (m, 1H), 0.86 (s, 9H).

EXAMPLE 11

H¹ (300 MHz CD₃OD), δ 8.56 (s br, 1H), 7.65 (t, J=6.6 Hz, 1H), 7.45 (t,J=7.2 Hz, 1H), 7.26 (t, J=7.8 Hz, 1H), 7.01 (d, J=8.7 Hz, 1H), 6.95 (d,J=6.0 Hz, 1H), 5.14 (d, J=10.5 Hz, 1H), 4.51 (d, J=10.5 Hz, 1H), 4.26(m, 1H), 3.55-3.50 (m, 2H), 3.37-3.31 (m, 3H), 2.08-2.02 (m, 1H),1.71-1.45 (m, 2H), 1.35-1.23 (m, 3H), 0.86 (s, 9H).

EXAMPLE 126-CHLORO-4′-(3-CHLORO-2-FLUORO-PHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID (3,4-DIHYDROXY-BUTYL)-AMIDE

H¹ (300 MHz CD₃OD), δ 8.55 (s br, 1H), 7.66 (t, J=6.6 Hz, 1H), 7.42 (t,J=7.8 Hz, 1H), 7.28-7.23 (m, 2H), 7.01 (d, J=8.7 Hz, 1H), 6.95 (d, J=6.0Hz, 1H), 5.20 (d, J=10.5 Hz, 1H), 4.53 (d, J=10.5 Hz, 1H), 4.26 (m, 1H),3.52 (m, 1H), 3.40-3.33 (m, 4H), 2.03 (m, 1H), 1.71-1.45 (m, 2H),1.35-1.28 (m, 1H), 0.86 (s, 9H).

EXAMPLE 136-CHLORO-4′-(3-CHLORO-PHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID (2-MORPHOLIN-4-YL-ETHYL)-AMIDE

¹H NMR (300 MHz, CD₃OH), δ 7.74 (d, 1H, J=7.5 Hz), 7.35-7.15 (m, 3H),7.11 (m, 1H), 6.88 (d, 1H, J=6.0 Hz), 5.22 (d, 1H, J=11.4 Hz), 4.55 (d,1H, J=9.6 Hz), 4.24 (d, 1H, J=6.3 Hz), 4.20-3.85 (m, 4H), 3.70-3.40 (m,4H), 3.25-3.00 (m, 4H), 2.09 (m, 1H), 1.27 (m, 1H), 0.934 (s, 9H).

EXAMPLE 146-CHLORO-4′-(3-CHLORO-PHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID (3-MORPHOLIN-4-YL-PROPYL)-AMIDE

¹H NMR (CD₃OD, 300 MHz), δ 7.72 (d, 1H, J=8.4 Hz), 7.38-7.22 (m, 3H),7.10 (m, 1H), 6.87 (m, 1H), 5.23 (d, 1H, J=11.1 Hz), 4.55 (d, J=6.9 Hz,1H), 4.28 (d, J=9.3 Hz, 1H), 4.20-3.85 (m, 4H), 3.70-3.40 (m, 2H),3.25-3.00 (m, 4H), 2.20-1.85 (m, 4H), 1.69 (d, J=15.3 Hz, 2H), 0.934 (s,9H).

EXAMPLE 156-CHLORO-4′-(3-CHLORO-PHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID (4,5-DIHYDROXY-PENTYL)-AMIDE

¹H NMR (CD₃OD, 300 MHz), δ 7.69 (d, 1H, J=8.4 Hz), 7.40-7.20 (m, 3H),7.10 (m, 1H), 6.86 (m, 1H), 5.24 (d, 1H, J=11.1 Hz), 4.51 (d, J=10.5 Hz,1H), 4.26 (m, 1H), 3.55-3.50 (m, 2H), 3.37-3.31 (m, 3H), 2.08-2.02 (m,2H), 1.71-1.45 (m, 2H), 1.35-1.23 (m, 3H), 0.86 (s, 9H).

EXAMPLE 166-CHLORO-4′-(3-CHLORO-PHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID (4-HYDROXY-3-HYDROXYMETHYL-BUTYL)-AMIDE

¹H NMR (300 MHz, CD₃OH), δ 7.69 (d, 1H, J=8.4 Hz), 7.40-7.20 (m, 3H),7.10 (m, 1H), 6.86 (m, 1H), 5.24 (d, 1H, J=11.1 Hz), 4.54 (d, J=10.5 Hz,1H), 4.34-4.31 (m, 1H), 3.51-3.49 (m, 4H), 3.36-3.32 (m, 2H), 2.08 (dd,J=15.3, 7.5 Hz, 1H), 1.53-1.49 (m, 2H), 1.33 (dd, J=15.3, 2.7 Hz, 1H),0.93-0.89 (m, 1H), 0.86 (s, 9H).

EXAMPLE 17(2′R,3S,3″S,4′R,5′R)6-CHLORO-4′-(3-CHLORO-4-FLUORO-PHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID (3,4-DIHYDROXY-BUTYL)-AMIDE

Formula: C₂₇H₃₁Cl₂F₂N₃O₄.HCl (HCl Salt) ¹H NMR (300 MHz, MeOD), δ 8.44(m, 1H), 7.70 (d, 1H, J=8.4 Hz), 7.41 (m, 1H), 7.14 (m, 2H), 6.90 (d,1H, J=6.0 Hz), 5.20 (d, 1H, J=11.1 Hz), 4.47 (d, 1H, J=8.1 Hz), 4.14 (d,1H, J=10.8 Hz), 3.47 (m, 4H), 1.91 (dd, 1H, J=15.6, 8.4 Hz), 1.57 (m,1H), 1.43 (m, 1H), 1.21 (dd, 1H, J=15.6, 1.8 Hz), 0.92 (s, 9H); ¹³C NMR(75 MHz, MeOD), δ 176.28, 166.52, 157.03, 153.19, 139.65, 130.67,129.58, 129.48, 128.46, 128.40, 124.54, 124.44, 122.58, 122.33, 121.41,121.17, 117.21, 116.93, 113.37, 113.03, 112.41, 69.69, 66.05, 64.36,62.93, 61.91, 55.48, 42.25, 36.99, 32.59, 29.84, 28.40.

EXAMPLE 18

EXAMPLE 196-CHLORO-4′-(3-CHLORO-4-FLUOROPHENYL)-2′-CYCLOPENTYLMETHYL-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID (3,4-DIHYDROXY-BUTYL)-AMIDE

¹H NMR (300 MHz, CD₃OD), δ 7.72 (d, 1H, J=8.5 Hz), 7.46-7.40 (m, 1H),7.23-7.12 (m, 2H), 6.88 (d, 1H, J=6.0 Hz), 5.12 (d, 1H, J=11.3 Hz),4.39-4.35 (m, 1H), 4.14 (d, 1H, J=11.3 Hz), 3.41-3.28 (m, 5H), 1.92-1.78(m, 2H), 1.62-1.42 (m, 8H), 1.28 (d, 1H, J=15.8 Hz), 1.10-0.90 (m, 2H).

EXAMPLE 20

EXAMPLE 21

EXAMPLE 226-CHLORO-4′-(3-CHLORO-4-FLUOROPHENYL)-2′-CYCLOHEXYL-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID (3,4-DIHYDROXY-BUTYL)-AMIDE

¹H NMR (300 MHz, CD₃OD), δ 8.54-8.40 (m, 2H), 7.56 (d, 1H, J=8.4 Hz),7.31-7.29 (m, 1H), 7.16-7.11 (m, 2H), 6.82 (d, 1H, J=6.1 Hz), 4.99-4.95(m, 1H), 4.21-4.01 (m, 2H), 3.39-3.19 (m, 5H), 2.50-2.40 (m, 1H),2.00-1.83 (m, 2H), 1.63-1.54 (m, 4H), 1.44-1.09 (m, 6H).

EXAMPLE 23

EXAMPLE 246-CHLORO-4′-(3-CHLORO-4-FLUOROPHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID [2-(3-HYDROXY-CYCLOPENTYL)-ETHYL]-AMIDE

¹H NMR (300 MHz, CD₃OD), δ 8.52-8.49 (m, 1H), 7.72 (d, 1H, J=8.5 Hz),7.40 (d, 1H, J=10.0 Hz), 7.16-7.15 (m, 2H), 6.90 (d, 1H, J=6.0 Hz),5.27-5.23 (m, 1H), 4.49 (d, 1H, J=8.2 Hz), 4.14-4.07 (m, 2H), 3.24-3.22(m, 1H), 3.10-3.02 (m, 1H), 1.93-1.83 (m, 2H), 1.75-1.18 (m, 7H),1.05-0.93 (m, 1H), 0.92 (s, 9H), 0.85-0.81 (m, 1H).

EXAMPLE 25

EXAMPLE 266-CHLORO-4′-(3-CHLORO-4-FLUOROPHENYL)-2′-CYCLOPENTYL-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID (3,4-DIHYDROXY-BUTYL)-AMIDE

¹H NMR (300 MHz, CD₃OD), δ 7.68 (d, 1H, J=8.5 Hz), 7.40-7.37 (m, 1H),7.16-7.11 (m, 2H), 6.86 (d, 1H, J=6.0 Hz), 5.15 (d, 1H, J=11.6 Hz), 4.27(d, 1H, J=11.0 Hz), 4.15 (d, 1H, J=11.6 Hz), 3.39-3.24 (m, 5H),2.30-2.28 (m, 1H), 2.10-2.05 (m, 1H), 1.62-1.31 (m, 7H), 1.15-1.12 (m,1H), 0.78-0.75 (m, 1H).

EXAMPLE 276-CHLORO-4′-(3-CHLORO-4-FLUOROPHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-5′-(4-METHYL-PIPERAZINE-1-CARBONYL)-1H-SPIRO[INDOLE-3,3′-PYRROLIDIN]-2-ONE

¹H NMR (300 MHz, CD₃OD), δ 7.73-7.70 (m, 1H), 7.58-7.51 (m, 1H),7.29-7.19 (m, 2H), 6.93-6.89 (m, 1H), 5.83-5.80 (m, 1H), 4.70-4.68 (m,1H), 4.44-4.25 (m, 2H), 3.98-3.52 (m, 3H), 3.36-3.22 (m, 2H), 2.92-2.85(m, 3H), 2.40-1.89 (m, 2H), 1.20 (d, 1H, J=15.3 Hz), 0.95-0.90 (m, 9H).

EXAMPLE 28

EXAMPLE 29

EXAMPLE 30

EXAMPLE 31

EXAMPLE 321′-ACETYL-6-CHLORO-4′-(3-CHLORO-4-FLUOROPHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID (3,4-DIHYDROXY-BUTYL)-AMIDE

¹H NMR (300 MHz, CD₃Cl), δ 8.08 (s, 1H), 7.19-7.12 (m, 1H), 6.93-6.77(m, 4H), 4.93 (d, 1H, J=10.2 Hz), 4.75 (d, 1H, 10.2 Hz), 4.00-3.98 (m,1H), 3.78-3.30 (m, 5H), 2.80-2.77 (m, 1H), 2.23 (s, 3H), 1.76-1.64 (m,1H), 1.56-1.52 (m, 2H), 0.92 (s, 9H).

EXAMPLE 336-CHLORO-4′-(3-CHLORO-4-FLUOROPHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1′-OXY-1,2,4′,5′-TETRAHYDRO-SPIRO[INDOLE-3,3′-PYRROLE]-5′-CARBOXYLICACID (3,4-DIHYDROXY-BUTYL)-AMIDE

¹H NMR (300 MHz, CD₃Cl), δ 7.35-7.27 (m, 1H), 7.09-7.07 (m, 1H),6.92-6.89 (m, 1H), 6.79-6.75 (m, 2H), 5.55 (d, 1H, J=11.0 Hz), 4.20 (d,1H, J=11.0 Hz), 3.67-3.65 (m, 2H), 3.44-3.32 (m, 3H), 2.80 (d, 1H,J=13.1 Hz), 1.80 (d, 1H, J=13.1 Hz), 1.70-1.60 (m, 2H), 0.84 (s, 9H).

EXAMPLE 34{[6-CHLORO-4′-(3-CHLORO-4-FLUOROPHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBONYL]-AMINO}-ACETICACID

¹H NMR (300 MHz, CD₃OD), δ 7.64 (d, 1H, J=8.4 Hz), 7.38 (d, 1H, J=6.2Hz), 7.27-7.21 (m, 1H), 7.10-7.07 (m, 1H), 6.90-6.84 (m, 1H), 5.08 (d,1H, J=9.9 Hz), 4.22-3.88 (m, 4H), 1.76-1.68 (m, 1H), 1.12-1.06 (m, 1H),0.84 (s, 9H).

EXAMPLE 35

EXAMPLE 366-CHLORO-4′-(3-CHLORO-4-FLUOROPHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID (2-IMIDAZOL-1-YL-ETHYL)-AMIDE

¹H NMR (300 MHz, CD₃OD), δ 8.99 (s, 1H), 7.78 (d, 1H, J=8.4 Hz),7.50-7.45 (m, 2H), 7.37-7.35 (m, 1H), 7.19-7.11 (m, 2H), 6.89 (d, 1H,J=6.0 Hz), 5.32 (d, 1H, J=11.4 Hz), 4.52 (d, 1H, J=6.8 Hz), 4.41-4.36(m, 2H), 4.19 (d, 1H, J=11.4 Hz), 3.73-3.31 (m, 2H), 1.99-1.91 (m, 1H),1.21-1.16 (m, 1H), 0.92 (s, 9H).

EXAMPLE 377-{[6-CHLORO-4′-(3-CHLORO-4-FLUOROPHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBONYL]-AMINO}-3,5-DIHYDROXY-HEPTANOICACID

¹H NMR (300 MHz, CD₃OD), δ 7.60 (d, 1H, J=8.4 Hz), 7.37 (d, 1H, J=7.5Hz), 7.08-7.06 (m, 2H), 6.87-6.84 (m, 1H), 4.84 (d, 1H, J=10.2 Hz),4.15-4.08 (m, 1H), 4.00-3.96 (m, 2H), 3.80-3.75 (m, 1H), 3.36-3.32 (m,2H), 2.49-2.43 (m, 2H), 1.65-1.55 (m, 6H), 1.04 (m, 1H), 0.92 (s, 9H).

EXAMPLE 386-CHLORO-4′-(3-CHLORO-4-FLUOROPHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID (2,3,4-TRIHYDROXY-BUTYL)-AMIDE

¹H NMR (300 MHz, CD₃OD), δ 7.83-7.66 (m, 1H), 7.30-7.10 (m, 3H),6.92-6.88 (m, 1H), 5.30-5.11 (m, 1H), 4.48-4.21 (m, 1H), 4.15-3.95 (m,1H), 3.64-3.60 (m, 2H), 3.51-3.47 (m, 2H), 3.32-3.24 (m, 2H), 1.95-1.88(m, 1H), 1.23-1.13 (m, 1H), 0.92-0.82 (m, 9H).

EXAMPLE 396-CHLORO-4′-(3-CHLORO-4-FLUOROPHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID (2-GUANIDINO-ETHYL)-AMIDE

¹H NMR (300 MHz, CD₃OD), δ 7.69 (d, 1H, J=8.4 Hz), 7.39 (d, 1H, J=6.7Hz), 7.14-7.04 (m, 2H), 6.89 (d, 1H, J=5.8 Hz), 5.24 (d, 1H, J=11.3 Hz),4.44 (d, 1H, J=8.0 Hz), 3.80-3.65 (m, 2H), 3.08-3.00 (m, 2H), 1.89-1.87(m, 1H), 1.71-1.64 (m, 2H), 1.40-1.33 (m, 1H), 0.91 (s, 9H).

EXAMPLE 40

EXAMPLE 416-CHLORO-4′-(3-CHLORO-4-FLUOROPHENYL)-5-FLUORO-2′-(2-METHYL-PROPENYL)-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID (3,4-DIHYDROXY-BUTYL)-AMIDE

¹H NMR (300 MHz, CD₃Cl), δ 8.19-8.11 (m, 1H), 7.28-7.19 (m, 3H),6.96-6.85 (m, 2H), 6.68 (d, 1H, J=5.9 Hz), 4.97 (d, 1H, J=8.9 Hz),4.60-4.55 (m, 1H), 4.30-4.27 (m, 1H), 3.82 (d, 1H, J=9.2 Hz), 3.71-3.23(m, 4H), 1.58-1.54 (m, 5H), 1.46 (s, 3H).

EXAMPLE 426-CHLORO-4′-(3-CHLORO-4-FLUOROPHENYL)-2′-(2,2-DIMETHYL-PROPYL)-5-FLUORO-2-OXO-1,2-DIHYDRO-SPIRO[INDOLE-3,3′-PYRROLIDINE]-5′-CARBOXYLICACID (2-UREIDO-ETHYL)-AMIDE

¹H NMR (300 MHz, CD₃OD), δ 7.71 (d, 1H, J=8.4 Hz), 7.43 (d, 1H, J=6.0Hz), 7.15-7.12 (m, 2H), 6.93-6.89 (m, 1H), 5.19 (d, 1H, J=11.2 Hz), 4.48(d, 1H, J=11.2 Hz), 4.16-4.13 (m, 1H), 3.32-3.23 (m, 2H), 2.97-2.89 (m,2H), 1.95-1.90 (m, 1H), 1.57-1.53 (m, 2H), 1.23-1.18 (m, 1H), 0.92 (s,9H).

EXAMPLE 43(2′R,3S,4′R,5′R)-6-CHLORO-N-((S)-3,4-DIHYDROXYBUTYL)-5-FLUORO-4′-(3-FLUOROPHENYL)-2′-NEOPENTYL-2-OXOSPIRO[INDOLINE-3,3′-PYRROLIDINE]-5′-CARBOXAMIDE

¹H NMR (300 MHz, CD₃OD), δ 8.43 (m, 1H), 7.72 (d, 1H, J=8.4 Hz),7.30-7.15 (m, 1H), 7.06 (d, 2H, J=8.88 Hz), 6.95 (d, 1H, J=7.8 Hz), 6.88(d, 1H, J=6.0 Hz), 5.28 (d, 1H, J=11.3 Hz), 4.51-4.40 (m, 1H), 4.16 (d,J=11.3 Hz), 3.50-3.20 (m, 5H), 1.93 (dd, 1H, J=8.3, 15.5 Hz), 1.62-1.36(m, 2H), 1.23-1.13 (m, 1H), 0.92 (s, 9H).

EXAMPLE 44(2′R,3S,4′S,5′R)-6-CHLORO-4′-(2,3-DIFLUOROPHENYL)-N-((S)-3,4-DIHYDROXYBUTYL)-5-FLUORO-2′-NEOPENTYL-2-OXOSPIRO[INDOLINE-3,3′-PYRROLIDINE]-5′-CARBOXAMIDE

¹H NMR (300 MHz, CD₃OD), δ 8.47 (m, 1H), 7.73 (d, 1H, J=7.2 Hz),7.43-7.30 (m, 1H), 7.23-7.15 (m, 2H), 6.89 (d, 1H, J=6.0 Hz), 5.29 (d,1H, J=11.1 Hz), 4.64 (d, 1H, J=11.4 Hz), 4.58-4.50 (m, 1H), 3.50-3.20(m, 5H), 1.93 (dd, 1H, J=8.3, 15.5 Hz), 1.67-1.36 (m, 2H), 1.23-1.13 (m,1H), 0.92 (s, 9H).

EXAMPLE 45(2′R,3S,4′R,5′R)-6-CHLORO-4′-(3,4-DIFLUOROPHENYL)-N-((S)-3,4-DIHYDROXYBUTYL)-5-FLUORO-2′-NEOPENTYL-2-OXOSPIRO[INDOLINE-3,3′-PYRROLIDINE]-5′-CARBOXAMIDE

¹H NMR (300 MHz, CD₃OD), δ 8.43 (m, 1H), 7.73-7.69 (m, 1H), 7.27-1.10(m, 2H), 6.97-6.94 (m, 1H), 6.90 (d, 1H, J=6.0 Hz), 5.26-5.23 (m, 1H),4.49 (d, 1H, J=7.5 Hz), 4.16-4.13 (m, 1H), 3.50-3.15 (m, 5H), 1.96-1.88(m, 1H), 1.62-1.40 (m, 2H), 1.23-1.13 (m, 1H), 0.92 (s, 9H).

EXAMPLE 46(2′R,3S,4′S,5′R)-6-CHLORO-4′-(2,5-DIFLUOROPHENYL)-N-((S)-3,4-DIHYDROXYBUTYL)-5-FLUORO-2′-NEOPENTYL-2-OXOSPIRO[INDOLINE-3,3′-PYRROLIDINE]-5′-CARBOXAMIDE

¹H NMR (300 MHz, CD₃OD), δ 8.49 (m, 1H), 7.71 (d, 1H, J=7.2 Hz),7.45-7.40 (m, 1H), 7.15-7.00 (m, 2H), 6.89 (d, 1H, J=6.0 Hz), 5.23 (d,1H, J=11.1 Hz), 4.63 (d, 1H, J=11.4 Hz), 4.58-4.50 (m, 1H), 3.50-3.20(m, 5H), 1.93 (dd, 1H, J=8.3, 15.5 Hz), 1.67-1.36 (m, 2H), 1.18 (dd, 1H,J=1.9, 15.5 Hz), 0.92 (s, 9H).

EXAMPLE 47(2′R,3S,4′S,5′R)-6-CHLORO-N-((S)-3,4-DIHYDROXYBUTYL)-5-FLUORO-4′-(2-FLUOROPHENYL)-2′-NEOPENTYL-2-OXOSPIRO[INDOLINE-3,3′-PYRROLIDINE]-5′-CARBOXAMIDE

¹H NMR (300 MHz, CD₃OD), δ 8.43 (m, 1H), 7.70-7.64 (m, 2H), 7.40-7.18(m, 2H), 7.01-6.94 (m, 1H), 6.85 (d, 1H, J=6.0 Hz), 5.25 (d, 1H, J=11.3Hz), 4.62 (d, 1H, J=12 Hz), 4.55-4.52 (m, 1H), 3.50-3.20 (m, 5H), 1.91(dd, 1H, J=8.3, 15.5 Hz), 1.62-1.32 (m, 2H), 1.18 (dd, 1H, J=1.8, 15.3Hz), 0.92 (s, 9H).

EXAMPLE 48(2′R,3S,4′R,5′R)-6-CHLORO-N-((S)-3,4-DIHYDROXYBUTYL)-5-FLUORO-4′-(4-FLUOROPHENYL)-2′-NEOPENTYL-2-OXOSPIRO[INDOLINE-3,3′-PYRROLIDINE]-5′-CARBOXAMIDE

¹H NMR (300 MHz, CD₃OD), δ 8.39 (m, 1H), 7.70 (d, 1H, J=8.5 Hz),7.25-7.21 (m, 2H), 7.03-6.96 (m, 2H), 6.87 (d, 1H, J=6.0 Hz), 5.26 (d,1H, J=11.3 Hz), 4.48 (m, 1H,), 4.12 (d, J=11.4 Hz), 3.50-3.20 (m, 5H),1.92 (dd, 1H, J=8.3, 15.5 Hz), 1.62-1.32 (m, 2H), 1.20 (dd, 1H, J=2.0,15.3 Hz), 0.92 (s, 9H).

EXAMPLE 49 Cell Growth Inhibition

One major advantage of non-peptide small-molecule inhibitors overpeptide-based inhibitors is their superior cell permeability. It ispredicted that potent, non-peptide inhibitors of the p53-MDM2interaction will be effective in inhibition of cell growth and divisionin cancer cells with a wild-type form of p53 through stimulation of theactivity of p53. Furthermore, they are predicted to have selectivity incancer cells with either a loss of p53 or a mutated, non-functional formof p53. To test these predictions, a cell growth assay was developedusing human prostate cancer LNCaP (p53 wild-type) and PC-3 (p53 null)cell lines. The toxic effects of compounds of the invention were tested.

Cells were seeded in 96-well flat bottom cell culture plates at adensity of 3-4×10³ cells/well and incubated in the presence of compoundsfor 4 days. The rate of cell growth inhibition after treatment withincreasing concentrations of the compounds was determined using WST-8(2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4disulfophenyl)-2H-tetrazoliummonosodium salt (Dojindo Molecular Technologies Inc., Gaithersburg,Md.). WST-8 was added at a final concentration of 10% to each well andthe plates were incubated at 37° C. for 2-3 hrs. The absorbance of thesamples was measured at 450 nm in a plate reader (Molecular Device-TECANULTRA). The concentration of the compounds that inhibited cell growth by50% (IC₅₀) was calculated by comparing absorbance in the untreated cellsand the cells treated with the compounds. As shown in Table 2, thecompounds of the invention were about 5-fold to about 50-fold moreeffective at inhibiting cell growth in p53-positive LNCaP cells ascompared to p53-negative PC3 cells.

The compounds of the invention were similarly tested against HCT116(wild-type p53), and HT-29 (mutant p53) colon cancer cell lines. Asshown in Table 2, the majority of the tested compounds exhibited ahigher potency for cell growth inhibition in p53-positive HCT116 cellsas compared to p53-negative HCT116 cells, with the largest difference inpotency being about 25-fold.

Since normal cells also have wild-type p53, a potential concern indevelopment of inhibitors of the p53-MDM2 interaction as new anti-cancerdrugs is that they may be nonselective and equally active in killingnormal cells as they are in killing cancer cells. Compounds will beevaluated in normal human prostate epithelial cells (PrEC) withwild-type p53 to confirm whether they display a good selectivity forcancer cells.

TABLE 2 IC₅₀ ± SD HCT116 HCT116 (μM)* LNCaP PC3 p53WT p53KO (Ki ± SD)IC₅₀ IC₅₀ IC₅₀ IC₅₀ Compound (μM) (μM) (μM) (μM) (μM) MDM2-319 0.0760.2738 15.4 0.7174 11.83 (0.0063) 0.1839 9.08 0.6145 12.2 0.077 0.22911.35 0.2466 14.23 (0.006) 0.2289 ± 0.04  11.61 ± 3.3 0.52 ± 0.24 12.75 ±1.29 0.064 (0.005) 0.092 0.115 0.122 0.13 0.18 0.17 0.16 0.10 MDM2-319-10.194 0.2612 HCT116 0.9085 12.66 0.072 0.3602 1.364 10.39 MDM2-319-2 1753.474 14.39 15.46 22.24 (16.43) 3.378 19.64 21.3 19.49 >80 4.005 19.588.508 25.44 3.61 ± 0.3  17.87 ± 3.01 15.08 ± 6.4  22.39 ± 2.97MDM2-319-3 0.37 1.758 16.65 3.813 13.84 (0.034) 1.241 17.45 4.109 24.780.45 0.8472 22.16 2.826 16.72 0.23 1.28 ± 0.4  18.75 ± 2.97  3.58 ± 0.6718.44 ± 5.67 MDM2-319-4 0.71 1.417 18.53 5.03 13.13 (0.066) 1.294 23.385.801 26.48 1.16 0.8902 25.6 2.413 13.39 0.46 1.21 ± 0.29  22.5 ± 3.61 4.41 ± 1.77 17.6 ± 7.6 MDM2-319-5 0.055 0.6020 9.47 0.9927 10.52 0.0400.4653 5.553 1.286 5.39 0.6341 8.711 1.502 8.403 0.56 ± 0.08 7.91 ± 2  1.23 ± 0.2  8.1 ± 2.5 MDM2-319-6 0.59 2.583 21.4 5.452 14.29 0.55 1.22320.83 7.41 16.06 Flu 1.931 14.15 5.957 14.58 1.91 ± 0.6  18.7 ± 4   6.26± 1.0 14.97 ± 0.9  MI-319-7 39.33 2.17 39.74 8.22 >30 1.917 34.297.954 >30 MI-319-8 0.34 0.9631 30.66 4.114 30.95 0.53 0.8928 20.72 4.73225.48 0.6625 18.43 2.738 22.69 1.46 ± 1.13 23.27 ± 6.5   3.86 ± 1.0226.3 ± 4.2 MI-319-9 3.58 2.866 20.91 7.48 9.711 2.7661.7 5.547 9.1988.189 5 6.09 4.631 4.52 2.46 ± 0.61 11.12 ± 8.5   7.1 ± 2.3  7.47 ± 2.66MI-319-10 0.22 0.7764 10.47 1.411 11.58 0.26 0.8283 10.36 1.947 9.840.4493 8.074 1.088 5.454 0.68 ± 0.2   9.63 ± 1.35 1.48 ± 0.4  8.9 ± 3.1MI-319-11 5.13 2.846 17.29 13.5 13.38 5.39 3.078 14.71 15.82 18 5.114.219 24.24 24.89 24.68 3.38 ± 0.73 18.7 ± 4.9 18.4 ± 5.9 18.85 ± 5.45MI-319-11-A 2.24 0.3078 1.765 2.153 3.966 1.64 0.5313 2.754 2.816 2.6270.4624 4.024 0.9753 4.182 0.43 ± 0.11 2.84 ± 1.1 1.98 ± 0.9  3.59 ± 0.84MI-319-12 2.30 1.974 22.35 12.37 13.45 2.96 2.571 20.89 13.2 17.84 1.93113.31 4.365 14.13 2.15 ± 0.35 18.85 ± 4.85  9.97 ± 4.87 15.14 ± 2.36MI-319-13 5.87 3.663 17.2 3.227 32.64 5.04 2.316 14.48 6.368 35.05 4.8520.28 10.98 33.02  3.6 ± 1.26 17.32 ± 2.9  6.85 ± 3.8 33.57 ± 1.29MI-319-14 2.04 4.221 40.08 8.008 >30 1.66 2.63 40.2 9.266 >30 1.979 >305.738 >30 2.94 ± 1.1  7.67 ± 1.7 MI-319-15 0.35 4.522 >30 12.13 >30 0.184.125 >30 12.89 >30 6.113 >30 13.89 >30 4.92 ± 1.05 12.97 ± 0.88MI-319-16 0.85 (Flu) 24.07 >30 >30 >30 0.76 (Flu) MI-319-17 0.39 1.09814 2.096 25.13 0.49 1.548 22.02 3.792 22.33 1.511 23.63 3.148 25.14MI-319-18 3.51 >30 >30 >30 >30 3.57 >30 >30 >30 >30 MI-319-19 0.50 1.23530.68 2.68 >30 0.49 2.398 >30 5.981 >30 1.2 41.65 4.596 77.64 MI-319-2027.44 >30 >30 >30 31.59 66.12 93.54 >100 MI-319-21 2.838 21.87 4.40211.76 1.836 14.07 3.884 13.25 Calculation of K_(i): MDM2 [10 nM]; F-6 [1nM]; New Sample MDM2 protein: K_(d): 0.88 ± 0.41 nM (Hyperbolicequation; using 1 nM 6F) K_(d): 2.22 ± 0.93 nM (Klotz plot; using 1 nM6F)

EXAMPLE 50 Effects of MDM2 Inhibitors on Expression of P53 and ItsTarget Gene Products MDM2 and P21

Cancer cells are treated with test compounds or 0.1% DMSO for 24 hours.Cells are harvested by trypsinization and washed with cold phosphatebuffer saline, pH 7.5 (Invitrogen, Carlsbad, Calif.). Cells are lysedfor 30 minutes in ice-cold lysis buffer (50 mM Tris (pH 7.5), 150 mMNaCl, 1 mM EDTA (pH 8.0), 25 mM sodium fluoride, 1% NP-40 and 0.1% SDS)containing 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluorideand protease inhibitor cocktail (Roche Applied Science, Indianapolis,Ind.). Next, cell extracts are centrifuged at 12,000×g at 4° C. for 10minutes to obtain clarified lysates. Protein is estimated by Bio-Raddye. Cell lysates containing 35 μg protein are resolved on a 4-20%tris-glycine gel (Invitrogen, Carlsbad, Calif.) and transferred ontopoly(vinylidene difluoride) membranes. Immunodetection of proteins onthe transfer membrane is performed by using anti-p53 (Ab-6, OncogeneResearch Products, Boston, Mass.), anti-MDM2 (SMP14, Santa CruzBiotechnology, Santa Cruz, Calif.) and anti-p21 (BD Biosciences, SanDiego, Calif.) mouse monoclonal antibodies. Antibody to β-actin (Sigma,St Louis, Mo.) is used to assess the protein loading. When RKO(wild-type p53) and HT-29 (mutant p53) colon cancer cell lines aretreated for 24 hours with the compounds of the invention, it is expectedthat the compounds will induce accumulation of p53 and its targetgene-products only in RKO cells expressing wild-type p53.

EXAMPLE 51 Cell Death and Apoptosis Induced by MDM2 Inhibitors

RKO (wild-type p53) and HT-29 (mutant p53) colon cancer cell lines andCCD-18Co normal colon fibroblast cells are treated with increasing dosesof the compounds of the invention for 4 days in 6-well Petri dishes.Trypan blue dye exclusion assays are performed to determine the abilityof the inhibitors to induce cell death. After 4 days of treatment,floating and adherent cells are harvested and stained with 0.2% oftrypan blue solution (Sigma, St Louis, Mo.). Each treatment is performedin triplicate and at least 100 cells are counted. Cells stained blue ormorphologically unhealthy cells are scored as dead cells. To evaluateapoptosis, sub-diploid DNA content in cells treated with or without testinhibitors is analyzed by propidium iodide (PI) staining. After washingonce with cold PBS, cells are fixed in 70% ethanol for 1 day at −20° C.Ethanol-fixed cells are then washed twice with PBS and stained with astaining solution containing propidium iodide (PI) at 50 μg/ml and RNAseA at 100 μg/ml in PBS, for 20 minutes in dark at room temperature.Acquisition of cells and analysis of sub-diploid DNA content isperformed by flow cytometry using CellQuest software. It is expectedthat only cancer cells expressing wild-type p53 will undergo apoptosisin response to administration of the compounds.

EXAMPLE 52 Effect of MDM2 Inhibitors on Cell Cycle Progression of ColonCancer Cells

Cell cycle progression is evaluated in RKO (wild-type p53) and HT-29(mutant p53) colon cancer cell lines and CCD-18Co normal colonfibroblast cells by determining S-phase cells by incorporation ofbromodeoxyuridine (BrdU) followed by staining with FITC-labeledanti-BrdU antibody and the total DNA-content by staining with7-aminoactinomycin D (7-AAD) according to manufacturer's instructions(BD Biosciences, San Jose, Calif.). Briefly, cancer and normal cells,after overnight incubation, are treated with or without test compoundsfor 22 hours, followed by an additional 2 hours of incubation with 10 μMof BrdU. Cells are harvested, fixed and stained with FITC-labeledanti-BrdU and 7-AAD. Cell cycle distribution is analyzed by flowcytometry. Cells are acquired and data analyzed by using CellQuestsoftware (BD Biosciences). It is expected that the compounds of theinvention will induce a dose-dependent depletion of S-phase in RKOcancer cells and in CCD-18Co normal colon fibroblast cells, both ofwhich express wild-type p53. It is also expected that the compounds willhave no appreciable effect on cell cycle progression of HT-29 cellsexpressing mutant p53.

EXAMPLE 53 Protection of Normal Cells Form Chemotherapy with MDM2Inhibitors

PrEC normal prostate epithelial cells are seeded in 6 well plates andincubated with compounds of the invention for 24 hours, then 1 μM TAXOL(paclitaxel) is added for 2 days. Trypan blue is used to determine cellviability. The data is expected to show that when normal prostateepithelial cells are pretreated with the compounds of the invention,cells are protected from TAXOL.

Having now fully described the invention, it will be understood by thoseof skill in the art that the same can be performed within a wide andequivalent range of conditions, formulations, and other parameterswithout affecting the scope of the invention or any embodiment thereof.All patents, patent applications and publications cited herein are fullyincorporated by reference herein in their entirety.

1. A compound having Formula I:

or a pharmaceutically acceptable salt thereof, wherein: X is N; Y is O,or S; R₁, R₂, R₄, R₅, R₆, and R₇ are independently H or optionallysubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl,heterocyclic, CO₂R′, OCOR′, CONR′R″, NR″COR′, NR′SO₂R″, SO₂NR′R″,(C═NR′)NR″R′″, or NR′R″; or R₇ forms an aryl, cycloalkyl, orheterocyclic group with one of R₅ or R₆; R₈ is H or optionallysubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl,heterocyclic, CO₂R′, OCOR′, CONR′R″, SO₂NR′R″, or (C═NR′)NR″R′″; eachR′, R″ and R′″ is independently H or optionally substituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic; or R′and R″, or R″ and R′″, form a ring; and R₃ is CONHR₁₁ wherein R₁₁ is anoptionally substituted cycloalkyl-alkyl or monoheterocycloalkyl-alkylgroup or a dihydroxyalkyl group not containing a hydroxyl group at the3-position of the alkyl group.
 2. The compound of claim 1, havingFormula II or Formula III:

or a pharmaceutically acceptable salt thereof.
 3. The compound of claim1, having Formula IV:

or a pharmaceutically acceptable salt thereof.
 4. The compound of claim3, having Formula V or Formula VI:

or a pharmaceutically acceptable salt thereof.
 5. The compound of claim1, having Formula VII:

or a pharmaceutically acceptable salt thereof.
 6. The compound of claim5, having Formula VIII or Formula IX:

or a pharmaceutically acceptable salt thereof.
 7. The compound of claim1, wherein one of R₁ and R₂ is optionally substituted aryl orheteroaryl.
 8. The compound of claim 1, wherein one of R₁ and R₂ iscycloalkyl, straight or branched alkyl, amide or ester.
 9. The compoundof claim 1, wherein one of R₅ and R₆ is C₃₋₁₈ alkyl, aryl, orheteroaryl.
 10. A compound having Formula X:

or a pharmaceutically acceptable salt thereof, wherein: R₁₀ is hydrogen,and R₁₁ is an optionally substituted cycloalkyl-alkyl ormonoheterocycloalkyl-alkyl group or a dihydroxyalkyl group notcontaining a hydroxyl group at the 3-position of the alkyl group; or R₁₀and R₁₁ together form an optionally substitutedmonocyclo-heterocycloalkyl group; R₁, R₅, and R₇ are independently H oroptionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, aryl, heterocyclic, CO₂R′, OCOR′, CONR′R″, NR″COR′, NR′SO₂R″,SO₂NR′R″, (C═NR′)NR″R′″, or NR′R″; or R₇ forms an aryl, cycloalkyl, orheterocyclic group with one of R₅ or R₆; R₈ is H or optionallysubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl,heterocyclic, CO₂R′, OCOR′, CONR′R″, SO₂NR′R″, or (C═NR′)NR″R′″; andeach R′, R″and R′″is independently H or optionally substituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic; orR′and R″, or R″and R′″, form a ring.
 11. The compound of claim 10,having one of Formulae XI-XXVI:

or a pharmaceutically acceptable salt thereof.
 12. The compound of claim10, having Formula XXVII or Formula XXVIII:

or a pharmaceutically acceptable salt thereof.
 13. A compound of claim 1selected from the group consisting of:


14. A compound selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
 15. A compound

or a pharmaceutically acceptable salt thereof.
 16. A pharmaceuticalcomposition comprising a compound of claims 1, 14, or 15 and apharmaceutically acceptable carrier.
 17. A method of treating a disorderin an animal, comprising administering to said animal a therapeuticallyeffective amount of a compound of claims 1, 14, or 15, wherein saiddisorder is selected from the group consisting of colon cancer andprostate cancer.
 18. The method of claim 17, further comprisingadministering an additional agent(s), wherein said additional agent(s)is selected from the group consisting of a chemotherapeutic agent andradiation.
 19. The method of claim 18, wherein said compound of claim 1is administered prior to said additional agent(s).
 20. The method ofclaim 18, wherein said compound of claim 1 is administered after saidadditional agent(s).
 21. The method of claim 18, wherein said compoundof claim 1 is administered concurrently with said additional agent(s).22. A kit comprising a compound of claims 1, 14, or 15 and instructionsfor administering said compound to an animal having cancer, wherein saidcancer is selected from the group consisting of prostate cancer andcolon cancer.
 23. The kit of claim 22, further comprising achemotherapeutic agent.
 24. A compound selected from the groupconsisting of:

or a pharmaceutically acceptable salt thereof, or a differentpharmaceutically acceptable salt thereof.
 25. A compound selected fromthe group consisting of:

or a pharmaceutically acceptable salt thereof, or a differentpharmaceutically acceptable salt thereof.